Interleukin-33 and Notch signaling in antiviral defense and inflammation

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and inflammation

by

Tor Espen Stav-Noraas

2018

Laboratory for Immunohistochemistry and Immunopathology Institute of Clinical Medicine, Faculty of Medicine

University of Oslo, Norway

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© Tor Espen Stav-Noraas, 2018

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8377-336-1

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.

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ACKNOWLEDGMENTS

This thesis is based on work carried out at the Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Institute of Pathology, University of Oslo, between August 2013 and September 2016. The project was financed by South-Eastern Norway Regional Health Authority, the Research Council of Norway and the University of Oslo.

I would like to express my sincere gratitude to my supervisor and head of the research group Professor Guttorm Haraldsen for giving me the chance to embark on the thesis work. You have been a great leader and supervisor for me during the period I stayed in your group. I would also like to thank my second supervisor Johanna Hol Fosse who has mentored me since I started as a PhD fellow. You have always encouraged me in my work and given me incentives and ideas for planning and executing experiments. I also thank my third supervisor Mari Kaarbø for introducing me to the world of virology, which has been indispensable for both my theoretical and practical work.

Reidunn Jetne Edelmann was a key contributor to this thesis as she did a lot of the initial work. I would like to thank you for letting me continue the work you started.

Furthermore, Lars La Cour Paulsen has been an important colleague and source of inspiration to me, teaching me a great deal about detailed planning and performing of experiments. In addition to being a great motivational source you have given me new ideas and inputs of how to solve challenges in experiments.

Thanks to Aaste Aursjø, Hege Eliassen, Marte Rabo Carlsen, Linda Solfjell, Kjersti Thorvaldsen, Dahn Phung, Filip Nikolaysen, Sara Halmøy Bakke and Kathrine Hagelsteen for their great work in maintaining the laboratory.

I would also like to thank past and present group members for being part of an inspiring work environment and taking part in scientific discussions.

Lastly, I thank my wife Hanne Kathrine Stav-Noraas for supporting me through this period.

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Abbreviations

Ad5 Adenovirus serotype 5 DSB Double strand break EC Endothelial cell

ISRE IFN-stimulated response element ILC Innate lymphoid cell

IFN Interferon

IRF Interferon regulatory factor ISG Interferon stimulated genes INF-γ Interferon-γ

IL Interleukin

KSHV Kaposi's sarcoma-associated herpesvirus LPS Lipopolysaccharide

MDA5 Melanoma Differentiation-Associated protein 5 MRN MRE11-RAD50-NBS1 complex

nrAd5 Non-replicative adenovirus serotype 5

NICD1 NOTCH1 intracellular domain, cleaved at Val1744 PAMP Pathogen associated molecular pattern

RBPJ Recombination signal binding protein for immunoglobulin kappa J region RIG-I Retinoic acid-inducible gene I

STAT Signal transducer and activator of transcription TLR Toll like receptor

TNF Tumor necrosis factor

VCAM-1 Vascular cell adhesion molecule-1 VEGF Vascular endothelial growth factor wtAd5 Wild type adenovirus serotype 5

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1 Papers Included

1.1 Paper I

Endothelial IL-33 Expression Is Augmented by Adenoviral Activation of the DNA Damage Machinery

Stav-Noraas TE, Edelmann RJ, Poulsen LLC, Sundnes O, Phung D, Küchler AM, Müller F, Kamen AA, Haraldsen G, Kaarbø M*, Hol J*

*) These authors contributed equally

J Immunol April 15, 2017, 198 (8) 3318-3325 1.2 Paper II

Endothelial IL-33 promotes ATM, pH2AX and p21 mediated DNA damage response

Stav-Noraas TE, Szymanska M, Edelmann RJ, Phung D, Renzi A, Poulsen L, Müller F, Haraldsen G, Kaarbø M*, Hol J*

*) These authors contributed equally Manuscript

1.3 Paper III

Inhibition of endothelial NOTCH1 signaling attenuates inflammation by reducing cytokine-mediated histone acetylation at inflammatory enhancers

Lars la Cour Poulsen¹, Reidunn Edelmann¹, Stig Krüger, Rodrigo Diéguez-Hurtado, Akshay Shah, Tor Espen Stav-Noraas, Monika Szymanska, Junbai Wang, Manuel, Ehling, Rui Benedito, Monika Kasprzycka, Espen Bækkevold, Olav Sundnes, Kim Midwood, Helge Scott, Philippe Collas, Christian Siebel, Ralf Adams, Guttorm Haraldsen, Eirik Sundlisæter², and Johanna Hol²

Arteriosclerosis, Thrombosis Vascular Biology. 2018:38:854-869 Originally published February 15.2018

1, 2) These authors contributed equally

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2 Introduction

2.1 General introduction

The inner layer of blood vessels is covered by endothelial cells that form an interface between circulating blood and the vessel wall (Potente, Gerhardt, & Carmeliet, 2011).

These vessels lead blood from the heart via arteries to the capillary bed for transport of nutrients and oxygen to cells and export of waste products before collecting to veins that return to the heart (Potente et al., 2011). In addition to transportation of metabolic compounds, endothelial cells are essential for vasoregulation, coagulation and cellular transportation in and out of tissues (Pober & Sessa, 2007; Vestweber, 2015). The endothelium is among the first line of defenses capable of detecting foreign pathogens and endogenous danger signals leading to production of pro-inflammatory cytokines and chemokines. Thus, endothelial cells function as sentinel cells mediating inflammation and immune cell mobilization (Mai, Virtue, Shen, Wang, & Yang, 2013).

2.2 Endothelial cells and vessel growth

In the embryo, new vessels form from mesoderm-derived endothelial precursors (angioblasts) that further differentiate into a primitive vascular labyrinth (vasculogenesis) and subsequently to vessel sprouts (angiogenesis) (Figure 1, A and B).

The endothelial cells that are attracted by proangiogenic signals will get motile, invasive and form protruding filopodia. These sprouting cells probe the environment for angiogenic signals and are called tip cells (Figure 1 B). Following these tip cells, stalk cells extend fewer filopodia and instead proliferate to support the sprouting elongation and subsequently form a lumen. To build vessel loops, tip cells form connections (anastomose) with cells from neighboring sprouts. The initiation of blood flow, establishment of a basement membrane and the stimuli from mural cells will contribute to stabilizing new connections. This sprouting process continues until proangiogenic signals are reduced, and then some of the sprouting networks will close up (Figure 1, C).

In zebrafish ventral sprouting is restrained by vascular endothelial growth factor (VEGF) and Notch (Potente et al., 2011). However, vascular endothelial growth factor-A

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promotes segregation of dorsal aorta and axial vein during development by binding to its receptor Kdrl (Kim et al., 2013). Endothelial cells and mural cells (vascular smooth muscle cells and pericytes) share a basement membrane composed of an extracellular matrix which confides endothelial tubules. At the onset of angiogenesis, endothelial cells need to be liberated from these physical constraints. Thus, proteolytic enzymes break up the basement membrane mediated by matrix metalloproteases (MMPs). One of these MMPs is the membrane type-matrix metalloprotease (MT-MMP1) which is enriched in tip cells. The enzymatic breakdown of the basement membrane also causes a release of angiogenic signals (Ucuzian, Gassman, East, & Greisler, 2010). On the other hand, to prevent inappropriate sprouting, proteolytic cleavage of the basement membrane also leads to formation of molecules capable of preventing endothelial cell migration and tube formation, thus inhibiting angiogenesis (Nyberg, Xie, & Kalluri, 2005). The proangiogenic part of the ECM is composed of intact molecules (etc. collagen, fibrillin and fibronectin). Enzymatic breakdown of these molecules can lead to generation of antiangiogenic fragments. As an example, the anti-angiogenic fragment arresten is derived from Collagen IV, and exerts its anti-angiogenic effect by inhibiting MAPK signaling (Neve, Cantatore, Maruotti, Corrado, & Ribatti, 2014). During vessel growth, the Notch ligand DLL4, expressed on tip cells, inhibits tip cell formation and is essential for establishing normal vasculature networks (Suchting et al., 2007). In addition, strong expression of Jagged 1 on stalk cells can antagonize DLL4 signaling from tip cell, thus promoting angiogenesis (Adams & Eichmann, 2010).

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Figure 1. Vessel growth. Angioblasts differentiate into endothelial cells (EC) which form cords. This leads to luminization, cord hollowing and differentiation into arterial or venous tubes (A). Activated endothelial cells differentiation into tip or stalk cells, this selection depends on lateral inhibition. These cells form guided sprouts (controlled by DLL4/Notch signaling), sprout fusion and lumen formation which can initiate blood flow (B). Sequential steps of vascular remodeling from primitive to a mature plexus (C).

(Potente et al., 2011). Used with permission.

2.2.1 The vascular barrier and maintenance of homeostasis

An intact layer of endothelial cells is important for maintaining homeostasis by regulating the fluctuation of liquid, macromolecules and cells. The semi-permeable barrier is enforced by lateral junctions of apposed endothelial cells, as well as focal adhesion points connecting the endothelium to the extracellular matrix. Among the most important intercellular barrier-forming structures are the adherens junctions, gap junctions and tight junctions. The increased vascular permeability that accompanies inflammation is mediated by controlled modification of the junction architecture.

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Disruption of the endothelial barrier can severely impact tissue function and viability.

Furthermore, increased vascular permeability is implicated in both acute and chronic diseases such as for example sepsis, stroke, dengue fever, malaria and arthritis. Thus, maintaining homeostatic conditions over the endothelial barrier is essential to prevent development of pathological conditions. Leukocytes contribute to regulating the increased vascular permeability accompanying a variety of pathological conditions. As an example, neutrophils are directly recruited to the site of injury and the release of reactive oxygen species, proteolytic enzymes and cytokines from neutrophils helps facilitate increased vascular leakage. Furthermore, interfering with neutrophil accumulation minimizes or prevents endothelial barrier dysfunction (Rodrigues &

Granger, 2015).

2.3 Immune system

Most or all of the cells in the human body take part in the defense against foreign pathogens imposing danger. The protective functions of the immune system range from barrier functions (skin and anti-microbial secretes (Schauber & Gallo, 2008)), recognition of pathogens by pattern recognition receptors, tissue repair mechanisms (wound seal by neutrophils swarming (Lammermann et al., 2013)), phagocytosis, complement activation, humoral responses and protective T-cell responses (Iwasaki & Medzhitov, 2015). The wide range immune functions are divided into the innate and adaptive (acquired) immune system. The adaptive immunity comprises cells adapting to new molecular structures by genomic recombination processes (B- and T-cells) and the innate immunity comprise cells which are genetically unchanged.

2.3.1 The role of macrophages

Macrophages have essential immune functions, such as clearing worn out cells and debris, viruses, bacteria, apoptotic cells and tumor cells. In addition, following injury and infection, macrophages can mount an inflammatory response and present antigens on MHC class II molecules to CD4 T+ cells. Furthermore, the macrophage is one of the most actively secreting cells, with the potential to secrete the pro-inflammatory cytokines IL- 1-β and TNF-α. These cytokines activate neighboring cells (e.g endothelial cells) and are essential in establishing a complete immune response. However, if the inflammatory

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response is not resolved, cytokine secretion can lead to cytotoxicity and tissue injury (Eichinger et al., 2015; Laskin, Sunil, Gardner, & Laskin, 2011; X. Zhang & Mosser, 2008).

2.3.2 Endothelial cells as sentinels

Endothelial cell innate immune function encompasses pathogen sensing and leukocyte recruitment (Mogensen, 2009). Endothelial cells express a range of different receptors that can recognize pathogen-associated molecular patterns and also host-derived damage-associated molecular patterns (Fitzner, Clauberg, Essmann, Liebmann, & Kolb- Bachofen, 2008; Rosin & Okusa, 2011). Some of the receptor classes which can sense bacterial and viral structures like RNA, DNA and surface molecules are Toll like receptors (TLR) (Takeda & Akira, 2015), RIG-I-like receptors (RLR) (Loo & Gale, 2011) and the Cyclic GMP-AMP synthase/2'-5'-Oligoadenylate synthetases (cGAS/OAS) family (Kranzusch, Lee, Berger, & Doudna, 2013). As an example of endothelial DNA sensing mechanism, Kaposi's sarcoma-associated herpesvirus (KSHV) infection of endothelial cells can lead to IFI-16 sensing of viral DNA and formation of inflammasomes (Kerur et al., 2011).

Furthermore, activation of these pathways can lead to IFN and proinflammatory cytokine production, which in turn can activate leukocyte recruitment (Sprague & Khalil, 2009). When bacteria and bacterial components like lipopolysaccharide (LPS) penetrate physical barriers, a coordinated series of events leads to recruitment of neutrophils.

TLR4 is expressed by sentinel immune cells (macrophages and mast cells) and can recognize LPS, leading to secretion of pro-inflammatory cytokines. However, endothelial cells express both TLR4 and receptors for pro-inflammatory cytokines (TNF-α and IL-1β), thus endothelial cells can respond to both bacterial compounds and to pro-inflammatory cytokines (Dauphinee & Karsan, 2006; Pober & Sessa, 2007). In fact, independent on sentinel immune cells recognition of LPS by the endothelium can lead to sequestration of neutrophils and clearance of an Escherichia coli infection (Andonegui et al., 2009).

Activation of endothelial cells, leading to leukocytes extravasation, depend on a multistep adhesion cascade. This model is based on the observations that leukocytes binds to endothelial cells by interacting with selectins and adhesion molecules (P- selectin, E-selectin and Intercellular Adhesion Molecule (ICAM) 1 and 2, and Vascular cell adhesion protein 1 (VCAM1). Thus, the leukocytes start rolling and slowing down, leading to arrest and crawling. Leukocytes continue by crossing the endothelial barrier

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either by paracellular diapedesis or transcellular diapedesis. The multistep adhesion cascade is facilitated by interaction with a range of different endothelial surface molecules, as designated in Figure 2 (Vestweber, 2015).

Figure 2. The multistep adhesion cascade. This figure shows the sequential events of capture, rolling, arrest and crawling of leukocytes depend on interaction with endothelial cells (Vestweber, 2015). Used with permission and modified.

2.3.3 Interleukin-1 cytokine family

There are currently 11 different members of the IL-1 family (IL1F1-11), seven of which function as receptor agonists (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β and IL-36γ), three as receptor antagonists (IL-1Ra, IL-36Ra, IL-38) and one as an anti-inflammatory cytokine (IL-37) (Figure 3). All cells of the innate immune system signal via or are affected by members the IL-1 family (Garlanda, Dinarello, & Mantovani, 2013; Sims & Smith, 2010).

2.3.3.1 Interleukin-1 receptor family

The ten members of the interleukin-1 receptor family consist of six receptor chains forming four signaling receptor complexes, two decoy receptors (IL1R2, IL18BP) and two negative regulators (the Toll/interleukin-1 receptor (TIR)8/SIGIRR and IL- 1RAcPb)(Garlanda et al., 2013). The extracellular portion of the receptors generally has three Ig-domains (except IL-18-binding protein (IL-18 BP) and TIR8, which have one Ig- domain). The intracellular portion is characterized by their TIR-domains necessary for interaction with Myd88 (Garlanda et al., 2013). This canonical TIR is shared with the TLR family (J. Wu & Chen, 2014). Hence, the TIR domains are critical in sensing microbes, tissue damage, innate immune activation and inflammation. The four signaling receptors

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are IL-1R (IL-1R1 and IL-1RAcP), IL-33R (ST2/IL33Rα and IL-1RAcP), IL-18R (IL-18Rα and IL- 18Rβ) and the IL-36R (IL-1Rrp2 and IL-1RAcP) (Garlanda et al., 2013) (Figure 3).

Figure 3. The interleukin-1 receptor and cytokine family. Here the IL-1 family members are schematically represented. The minus sign represents negative regulators. The TIR domain of the TIR8 (SIGIRR) receptor has two amino acid substitutions (Cys222 and Leu305 for the canonical Ser447 and Tyr536) possibly consistent with non-conventional signaling. (Garlanda et al., 2013). Modified and used with permission.

2.3.3.2 Interleukin-1

IL-1 can affect most of the cells in the human body and is a mediator of autoinflammatory (e.g. rheumatid arthritis), autoimmune (Sjögren’s syndrome), infectious and degenerative disease (Lopalco et al., 2015). IL-1α and IL-1β are two distinct genes coding for proteins with similar biological properties, both capable of binding and activating IL-1R. However, while IL-1α is present in the epithelial layer of the entire gastro intestinal tract, lung, liver, kidney, endothelial cells and astrocytes (Garlanda et al., 2013) and is released upon cellular damage leading to necrosis (as occurs in ischemic diseases), IL-1β is very carefully regulated and requires activation before release. Nevertheless, IL-1α and IL-1β both function as “alarmins”. Following hypoxia IL-1α, but not IL-1β, is upregulated and subsequently released from dying cells.

The release of IL-1α is correlated with the recruitment of neutrophils, and later the release of mature IL-1β is correlated with the recruitment of macrophages.

Furthermore, IL-1α was shown to be capable of recruiting neutrophils and IL-1β for recruiting monocytes/macrophages (Rider et al., 2011). Thus, IL-1α accounts for the

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early phase of sterile inflammation and IL-1β for the later phase of sterile inflammation (Rider et al., 2011).

2.3.4 Sensing infections - Recognition of pathogen-associated molecular patterns

Microbial and viral pathogens can be sensed directly because they carry/contain pathogen-associated molecular patterns (PAMPs) or indirectly as virulence factors.

Pathogen-associated molecular patterns are conserved structures present on all microorganisms of a given class (both pathogenic and commensal) which can be recognized by pattern-recognition receptors (Medzhitov, 2008; Rakoff-Nahoum, Paglino, Eslami-Varzaneh, Edberg, & Medzhitov, 2004). Among the pattern recognition receptors, Toll like receptors (TLRs) have been studied extensively. These receptors are functionally diverse and can function as lipid sensors (TLR-1, -2, -4, -6) or nucleic acid sensors (TLR-3, -7, -8, -9) as well as sensor of other structures (Mogensen, 2009) Examples of other pattern recognition receptors are DNA sensors (cGAS (Sun, Wu, Du, Chen, & Chen, 2013), MRE11 (Kondo et al., 2013)) and RNA sensors (RIG-I and MDA-5 (Jensen &

Thomsen, 2012; J. Wu & Chen, 2014; Yoneyama et al., 2004). An overview of sensing and signaling by the DNA sensors cGAS and the RNA sensors RIG-I, MDA5 and LGP2 are outlined in Figure 4 (Radoshevich & Dussurget, 2016). MRE11 is more commonly described as a sensor of double stranded breaks to the DNA, which is described in chapter 2.7.1.

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Figure 4. Schematic overview of the DNA sensors cGAS, RIG-I, MDA5 and LGP2 sensing and signaling. The DNA sensor cGAS recognizes DNA leading to formation of cGAMP and subsequently STING-TBK-1-IRF-3 signaling. In addition, 5’pppdsRNA sensed by RIG-I and long dsRNA are sensed by MDA5 and LGP2. Activation of these RNA sensors leads to signaling mediated through CARD domains in the mitochondrial membrane. Activation of TBK1 and IRF-3 can then initiate a type 1 IFN response. STING (stimulator of interferon genes); cGAS (cyclic GMP-AMP Synthase); cGAMP (cyclic guanin-adenin monophosphate); TBK-1 (TANK-binding kinase 1); IRF-3 (Interferon regulatory factor-3);

MDA5 (Melanoma Differentiation-Associated protein 5) and LGP2 (Laboratory of Genetics and Physiology 2); CARD (Caspase recruitment domains) (Radoshevich &

Dussurget, 2016)). Used with permission.

2.3.5 Sensing damage – Recognition of Damage-Associated Molecular Patterns and alarmins

After cellular injury, damage-associated molecular patterns (DAMPs) leak out of the damaged cells and can be recognized by pattern recognition receptors. DAMPs include heat shock proteins, ATP, nucleosomes, mitochondrial components and many alarmins.

Alarmins are a group of proteins with intracellular and extracellular proporties that can either exert beneficial housekeeping functions (leading to tissue repair) or induce inflammation. This group of proteins shares conserved regulatory mechanisms, as the: 1.

secretory route, 2. post translational modifications and 3. proteolytic cleavage, which alter their function in time and space. Alarmin members consist of, among others, HMGB1, IL-1α, IL-33 and S100 (Bertheloot & Latz, 2017). HMGB1, IL-1α and IL-33 are nuclear proteins that all lack a secretion signal, thus active release might depend on

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non-canonical secretory vesicles. Furthermore, upon passive release by necrotic cells these alarmins promote inflammation and recruitment of leukocytes (Bertheloot & Latz, 2017). IL-33 is described in detail in chapter 2.5.

HMGB1 can be released either passively by necrotic and damaged cells or by mechanisms triggered upon immune cell activation. Once released, HMGB1 mediates inflammation, cell migration, proliferation and differentiation. The functional properties of extracellular HMGB1 depend on its redox state. As an example, formation of a cysteine bond between Cys23 and Cys45 confers pro-inflammatory properties to HMGB1. Furthermore, reduced HMGB1 (with all cysteines in thiol state) loses its alarmin function and instead behaves as a chemoattractant (Venereau et al., 2012). In addition, intracellular HMGB1 can actively be secreted by macrophages and monocytes stimulated with interferon-γ (INF-γ), TNF-α, IL-1β and LPS.

IL-1α is constitutively expressed in most resting cells and IL-1α plays an important role in inflammation induced by necrosis or tissue damage following ischemia. Moreover, in peritonitis, IL-1α from necrotic cells promotes neutrophil recruitment and peritoneal inflammation by binding to IL1R1 present on mesothelial cells (Bertheloot & Latz, 2017).

This was described in more detail under chapter 2.3.3.2.

2.4 Inflammation

Inflammation is the body’s immediate response to infection, danger or damage, and leads to recruitment of leukocytes from the blood to the tissues with the aim to eliminate invading pathogens and initiate repair. Tissue damage, pathogenic infections, allergy or autoimmune responses can all initiate inflammation (Carthew & Sontheimer, 2009; Lopalco et al., 2015; Medzhitov, 2008). There are five cardinal signs of inflammation: redness, heat, swelling, pain and reduced function. These are caused by increased blood flow (redness and heat), localized leakage of plasma protein-rich fluid into the tissue (swelling) and mediators released from leukocytes on C-type sensory nerve fibers (pain). Loss of function is caused by a combination of these factors (Pober &

Sessa, 2007). During the initial phase of inflammation, cells of the innate immune system are activated by recognition of molecular patterns from exogenous or endogenous

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sources. The initial recognition of infection is mediated by tissue-resident macrophages and mast cells that upon activation respond by producing chemokines, cytokines and vasoactive amines (Medzhitov, 2008). The main effect of these mediators is to induce an immediate and selective recruitment of leukocytes (mainly neutrophils) and plasma proteins from the blood. This is facilitated by activation of endothelial cells in post- capillary venules leading to selective extravasation (Pober & Sessa, 2007). Neutrophils are the main cell type among the leukocytes recruited during the early phase of infection. When the neutrophils reach the afflicted site, they are activated either by cytokines (secreted by tissue resident cells) or due to direct contact with pathogens. The neutrophil can kill the invading pathogens by releasing toxic components, like reactive oxygen species (ROS), reactive nitrogen species, proteinase 3, cathepsin G and elastase.

Thus, neutrophils also mediate tissue damage in the attempt to clear the infection (Nathan, 2006). A successful inflammatory response to an infection leads to elimination of the pathogen and tissue repair (Medzhitov, 2008). Tissue repair is mainly resolved by tissue resident and recruited macrophages (Serhan & Savill, 2005). The switch from pro- inflammatory prostaglandins to lipoxins leads to monocyte recruitment and is crucial for resolving inflammation and tissue damage as monocytes can remove dead cells and initiate tissue remodeling (Serhan & Savill, 2005). Resolvins, protectins and transforming growth factor-β (TGF-β) also play important roles in resolving inflammation (Chiurchiu et al., 2016; Serhan & Savill, 2005). In cases where the infection is not cleared quickly by the innate immune system, the adaptive immune system (B- and T-cells) will be activated. And if the infection still persists, formation of granulomas and tertiary lymphoid organs can occur (Drayton, Liao, Mounzer, & Ruddle, 2006).

2.4.1 Signaling in inflammation

Inflammatory signaling through pattern recognition receptors can activate a diverse range of transcription factors (RELA, Nuclear Factor Kappa B (NF-κB), AP1 and IRF) via signaling cascades consisting of among others Myd88, IKKy, IkBα, IRAK, ERK1/2 and p38 (Newton & Dixit, 2012).

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As an example of inflammatory signaling, binding of LPS to TLR4, of TNF-α to TNF-R or IL- 1β to IL-1R all induce canonical NF-κB signaling. This leads to phosphorylation and activation of IKKβ which then phosphorylates IkBα (pIkBα). While unphosphorylated IkBα binds NF-κB dimers preventing signal transduction, phosphorylation of IkBα leads to polyubiquitination and degradation by proteosomes. NF-κB homo- or heterodimers are then free to translocate to the nucleus and bind chromatin. NF-κB signaling can also be non-canonical or non-typical as displayed in Figure 5.

2.4.1.1 NF-κB transcription factor

The transcription factor NF-κB is a dimeric complex that can be composed by five subunits: RELA (p65), RELB, C-REL, NF-κB1 (p105) and NF-κB2 (p100). In contrast to the other subunits, NF-κB1 and 2 are synthesized as pro-forms (p105 and p100) which are processed to their shorter forms p50 and p51. All members of the NF-κB complex harbor an N-terminal REL homology domain which facilitates DNA contact and homo- and hetero-dimerization. In addition, three of the members (RELA, RELB and C-REL) contain C-terminal transactivation domains necessary for transcriptional activity. NF-κB signaling is mediated by the IkB family of proteins and the IkB kinase complex. The IkB family of proteins consists of four members: IkBα, IkBβ, IkBε and BCL-3. These members have ankyrin repeats which facilitate binding to NF-κB family proteins. And the three most important IkB kinase complexes are NEMO (NF-κB Essential Modulator), IKKα and IKKβ.

Furthermore, NF-κB crosstalks with other signaling mediators, such as kinases (GSK3-β, p38 and PI3K), transcription factors (STAT3 and p53), miRNAs and reactive oxygen species (Hoesel & Schmid, 2013; Lawrence, 2009). In addition to the signaling pathways described above, other pattern recognition receptors such as the DNA sensors TLR9 (Schoggins & Rice, 2011) and DNA-PK (Ferguson, Mansur, Peters, Ren, & Smith, 2012), as well as the DNA sensor signaling mediator STING can induce NF-κB signaling (Abe &

Barber, 2014).

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Figure 5. Canonical and non-canonical NF-κB signaling. In the canonical signaling pathway, LPS, TNF-α and IL-1β bind to their respective receptors. This leads to activation of the IKKβ complex, which phosphorylates IkB on s32 and s36. Phosphorylation of IkB leads to polyubiqutination and proteasomal degradation, thus dissociation from NF-κB.

This enables NF-κB to translocate to the nucleus and bind to NF-κB promoters (A). In the non-canonical signaling pathway, activation of BAFFR (B Cell activation factor), CD40, RANK (Receptor activator for NF-κB) or LTβR (lymphoxin B-receptor) leads to activation of IKKα by NIK (NF-κB-inducing kinase). IKKα can further phosphorylate P100 on S866 and S870, which leads to polyubiquitination of P100 and consequently proteosomal processing to p52.p52 RELB heterodimers. P52.p52 RELB can then translocate to the nucleus and bind NF-κB promoters. In atypical NF-κB signaling, genotoxic stress leads to translocation of NEMO into the nucleus where it is sumoylated and subsequently ubiqutinated. This process is mediated by ATM (ataxia telangiectasis mutated). ATM and NEMO can then translocate to the cytosol and activate IKKβ (Hoesel & Schmid, 2013), used with permission.

2.4.1.2 Interferon regulatory factors

The family of human interferon regulatory factors (IRF) consists of nine members (IRF-1 to 9) (Fujita et al., 1988; Tamura, Yanai, Savitsky, & Taniguchi, 2008). Each of these IRFs contains a conserved, 120 amino acid, N-terminal DNA binding domain (DBD) with five conserved tryptophan-rich repeats (Yanai, Negishi, & Taniguchi, 2012). The DNA binding domain forms a helix-turn-helix structure and recognizes the IFN-stimulated response element (ISRE) in the promoter region of the interferon-stimulated genes (Darnell, Kerr,

& Stark, 1994). Furthermore, IRF-1 also binds to the positive regulatory domain I (PRD-I) which is involved in the IFN-β response to dsRNA. As an example of different

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functionality within the IRF family, IRF-3 does not bind to the PRD-I domain. (Au, Moore, Lowther, Juang, & Pitha, 1995a; Whiteside, Visvanathan, & Goodbourn, 1992). ISREs are characterized by the consensus sequence “AANNGAAA” (“N” indicates any base out of Adenine (A), Cytosine (C), Guanine (G) or Thymidine (T)) (Fujii et al., 1999). Activation of IFN receptors activates STATs, which further induces phosphorylation and activation of IRFs leading to translocation and binding to ISREs (Au, Moore, Lowther, Juang, & Pitha, 1995b; C. Wu et al., 2015). This in turn leads to activation of a range of different ISGs by IFN-α, -β and -γ (each induces a unique set of ISGs) (Der, Zhou, Williams, & Silverman, 1998). Many viruses can also trigger activation of ISGs involved in preventing viral replication (Schoggins & Rice, 2011). In relation to this, IL-33 expression can be stimulated in macrophages by the Newcastle Disease Virus (NDV) in an IRF-3-dependent manner (Polumuri et al., 2012). In the following paragraphs, I will further focus on IRF-1 and -3 which have been targeted for discussion in the papers included in this thesis.

2.4.1.2.1 IRF-1

IRF-1 is an intrinsic antiviral factor involved in inhibiting replication of a broad range of viruses by inhibiting translation of viral effector genes (Schoggins et al., 2014; Schoggins et al., 2011). There is a close relationship between viral infection and host-cell DNA response. Viruses can selectively activate or repress the host-cell DNA damage response and viral replication can both be promoted and inhibited by host-cell DNA damage response (Turnell & Grand, 2012). Therefore, it is interesting that IRF-1 is involved in many aspects of the DNA damage response (DDR). For example, upon DNA-damage induced by exposure to the chemotherapeutic etoposide or irradiation, the expression of IRF-1 is induced and the half-life increased (Pamment, Ramsay, Kelleher, Dornan, &

Ball, 2002). Furthermore, cells depleted of the DNA damage response sensor ATM failed to induce IRF-1 in response to genotoxic stress; however, the IRF-1 response to viral mimetics stayed intact. In addition, IRF-1 and the DNA damage response protein P53 cooperated to induce P21 expression in Human colon cancer cells (HCT116) (Pamment et al., 2002). A ChIP-chip (Chromatin immune precipitation-chip) analysis of IRF-1 revealed new binding partners, and one of the major functional categories was the DNA damage response pathway. Furthermore, IRF-1 was shown to positively regulate the DNA repair protein BRIP1 and cells lacking IRF-1 were hypersensitive to the DNA

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crosslinking agent melphalan (Frontini, Vijayakumar, Garvin, & Clarke, 2009). Thus, IRF-1 seems to play a central role in DNA repair mechanisms.

2.4.1.2.2 IRF-3

IRF-3 is best known as a transcription factor mediating type 1 interferon signaling. IRF-3 is essential for mediating a virus-induced IFN response (Honda & Taniguchi, 2006; Sato et al., 2000) and is phosphorylated upon activation of a range of different pattern recognition receptors (cGAS, RIG-I/MDA5 and DAI) (Sun et al., 2013; Tamura et al., 2008)) recognizing DNA and RNA. DNA-PK is another DNA sensor (commonly associated to DNA repair) activating IRF-3 in a STING/TBK1 dependent manner (Ferguson et al., 2012).

2.4.1.3 Activation of inflammatory enhancer regions

Pro-inflammatory stimuli elicit a rapid response facilitated by alteration of the chromatin structure and binding of transcription factors to enhancer regions (Kaikkonen et al., 2013). Following activation of the NF-κB signaling pathway, the NF-κB transcription factor binds to DNA cis-regulatory elements at enhancers and promoters, thus promoting inflammatory signaling (Pierce, Lenardo, & Baltimore, 1988). Endothelial cells exposed to pro-inflammatory TNF-α rapidly deploy NF-κB to enhancers and promoters genome-wide. Furthermore, NF-κB recruits BRD4 (bromodomain-containing protein 4) which facilitates the establishment of new super enhancer regions (Brown et al., 2014).

Super enhancers are massive enhancer regions which concentrate chromatin-bound co- activators to genes essential for specialized cellular function (loven et al 2013). The ability of transcription factors to activate transcription depends on the recruitment of co-activators, which often have histone acetyl transferase (HAT) activity. Two such co- activators with histone acetyl transferase activity are P300 and CREB-binding proteins (CBP). Both P300 and CBP are ubiquitously expressed and are recruited (often together) by a broad range of sequence-dependent activators representing distinct transcription factors. Thus, they are well suited for enhancer mapping in different cell types and tissue. Nonetheless, a subset of regulatory elements depends on co-factors other than P300 and CBP. Therefore, enhancer mapping by measuring CBP and P300 chromatin binding will not cover all enhancer activity. One of the main substrates acetylated by P300 and CBP is lysine 27 of histone protein 3 (H3K27). The presence of H3K27

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acetylation (H3K27ac) is commonly associated with active enhancer regions, similarly to P300 and BCP. Thus, H3K27ac is used as a marker for measuring enhancer activity, and is considered more specific than P300 (Calo & Wysocka, 2013)

2.4.2 Endothelial cells in inflammation

Resting endothelial cells do not interact with leukocytes. This is because they sequester leukocyte interactive proteins such as P-selectin and chemokines within Weibel-palade bodies. In addition, resting endothelial cells limit expression of adhesion molecules as VCAM1, E-selectin and Intercellular Adhesion Molecule 1 (ICAM1) (Pober & Sessa, 2007).

During acute inflammation, endothelial cells are activated, leading to the recruitment of neutrophils into the tissue. The majority of leukocytes crosses the endothelial cell barrier by passing between endothelial cells (Marchesi, 1961). The adhesion molecules Platelet endothelial cell adhesion molecule (PECAM)(CD31) and MIC2 (CD99) are present at adherent and tight junctions between endothelial cells. Both PECAM and MIC2 participate in interactions with neutrophils and monocytes, facilitating transmigration across the endothelial lining (Schenkel, Mamdouh, Chen, Liebman, & Muller, 2002). This is also described in chapter 2.3.2 “Endothelial cells as sentinels”. In addition to crossing the intra-endothelial barrier, leukocytes can pass through endothelial cells (Feng, Nagy, Pyne, Dvorak, & Dvorak, 1998). As shown in figure 6, stimulation of endothelial cells with the inflammatory cytokines IL-1 and TNF (tumor necrosis factor) (secreted by leukocytes) activates the transcription factors NF-κB subunit and Activator Protein 1 (AP1). This leads to increased expression of E-selectin, ICAM1, VCAM1, chemokines and cyclooxygenase 2 (COX2) (Pober & Sessa, 2007). The inflammatory stimuli can cause increased prostaglandin I2 (PGI2) due to increased COX2. PGI2 is a potent vasodilator and inhibitor of platelet aggregation. The inflammatory stimulus also leads to activation of leukocyte adhesion and proliferation of vascular smooth muscle cells (Ricciotti &

FitzGerald, 2011). Additionally, IL-1 and TNF stimulation of endothelial cells triggers reorganization of the actin and tubulin cytoskeleton so that gaps between endothelial cells can form (Petrache, Birukova, Ramirez, Garcia, & Verin, 2003; Pober et al., 1987).

Hence, inflammatory activation (IL-1 and TNF) of endothelial cells leads to increased blood flow, increased vascular leakage of plasma proteins and increased leukocyte recruitment at the affected site.

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Figure 6. Inflammatory activation of endothelial cells. In response to inflammatory cytokines (as TNF or IL-1), signaling by NF-κB and activating protein 1 (AP1) is activated by upstream signaling mediators as designated in the figure. This leads to transcriptional activation of E-selectin, ICAM1, VCAM1, chemokines and COX2. COX2 further leads to increased levels of prostaglandin H2 (PGI2)(Pober & Sessa, 2007). Used with permission.

2.5 Adenovirus

Adenovirus capsid proteins and viral genetic material can be recognized by the host, leading to inflammation and can therefore be characterized as PAMPs (Chintakuntlawar, Zhou, Rajaiya, & Chodosh, 2010). Adenovirus was discovered in adenoid cell cultures by Rowe et al in 1953 (Rowe, Huebner, Gilmore, Parrott, & Ward, 1953), and its icosahedral structure was published by Horne et al in 1959 (Horne,

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Brenner, Waterson, & Wildy, 1959). The Adenoviridae family consists of five genera;

Ichtadenovirus, Mastadenovirus, Aviadenovirus, Atadenovirus and Siadenovirus (Harrach et al., 2011). Furthermore, currently 52 human serotypes (Jones et al., 2007) have been recognized, these are subdivided into six groups, from A-F (Russell, 2009).

The vectors used in this thesis are derived from adenovirus serotype 5 (Ad5) which belong to the Mastadenovirus family. Ad5 is a non-enveloped double stranded DNA virus with a 36 kilo base linear genome consisting of four early transcriptional units 1-4 (E1-E4), and five late transcriptional units (L1-L5) (Davison, 2003; Hendrickx et al., 2014;

Tatsis & Ertl, 2004). Non-replicative versions of the adenovirus designed for gene delivery are classified into first generation (Ad5ΔE1 and Ad5ΔE1ΔE3, lacking transcriptional unit E1 with or without E3, respectively), second generation (Ad5ΔE1ΔE3ΔE4, lacking units E1, E3 and E4) and helper dependent adenovirus (deprived of all adenoviral genes) (Alba, Bosch, & Chillon, 2005; Parks et al., 1996) However, helper dependent adenovirus still contains the inverted terminal repeats (ITR) and the packaging signal (ψ) essential for viral DNA replication (Figure 7). The first generation adenovirus is widely used as a laboratory tool to overexpress or disrupt protein synthesis and is considered to be a promising candidate for therapeutic gene delivery. Indeed, several adenoviral vectors have been approved for clinical trials (Ginn, Alexander, Edelstein, Abedi, & Wixon, 2013; Roth, 2006) and a non-replicative adenovirus containing the p53 gene is already in use to treat neck and head carcinomas (Peng, 2005).

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Figure 7. Genomic structure of wild type, first, second and helper dependent adenovirus serotype 5 (Ad5). Wild type Ad5 sequences are labelled in black, with the localisation of early genes 1-4 represented by arrows. Deleted segments are shown as thin lines or striped boxes (for alternative deletion). ITR: inverted terminal repeats; Ψ:

packaging signal. (Kosinska, Zhang, Lu, & Roggendorf, 2010). Used with permission.

2.5.1.1 Sensing of Adenovirus

Upon entering the host cell, the adenovirus binds to the coxsackie- and adenovirus receptor (CAR) and to integrins, initiating clathrin-dependent endocytosis. The adenovirus disrupts the endosome and travels via the microtubule to the nucleus entering by binding to histones (Gastaldelli et al., 2008; Meier & Greber, 2004). On this path through the cell, the adenovirus can interact with a range of host proteins (Figure 8), either by binding to viral proteins or viral DNA (Hendrickx et al., 2014). The toll like receptor 9 is an endosomal DNA sensor expressed by endothelial cells capable of sensing adenoviral DNA leading to activation of NF-κB (Schoggins & Rice, 2011). cGAS is another DNA sensor that can recognize adenoviral DNA (involving STING, TBK-1, IRF-3 (Nociari, Ocheretina, Schoggins, & Falck-Pedersen, 2007) activating STAT1 (Der et al., 1998; Lam, Stein, & Falck-Pedersen, 2014). Furthermore, the adenoviral DNA can be sensed by the DNA damage sensor MRE11-RAD50-NBS1 (MRN) complex activating a DNA damage response (Stracker, Carson, & Weitzman, 2002). In addition to DNA sensing mechanisms, sensing of adenovirus capsid proteins can lead to induction of keratitis in mice (Chintakuntlawar et al., 2010). To overcome immune regulation by the host, the adenovirus contains genes and proteins capable of restricting these interactions.

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Figure 8. Schematic representation of human Adenovirus (A), its route of infection and potential subcellular sensors (B).

Human adenovirus components can be divided into large capsid proteins, small capsid proteins and core proteins (B). The viral infection route consists of receptor mediated endocytosis, cytoplasmic transport, nuclear import of viral DNA, transcription and viral DNA replication. During this route, adenovirus can be recognized by endogenous sensors (Hendrickx et al., 2014). Although the nucleus is marked as a question mark, nuclear sensors recognising adenovirus DNA exists (IFI-16) (Kerur et al., 2011).

Used with permission

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2.6.1 IL-33 regulation

The human IL-33 gene is encoded on the short arm of chromosome 9, originally reported to contain seven exons (Baekkevold et al., 2003). Mouse IL-33 is encoded on chromosome 19 (Baekkevold et al., 2003; Schmitz et al., 2005) consisting of 11 introns and eight exons transcribed into seven different mRNA species, with five alternatively spliced and two unspliced versions (Figure 9A)(N. T. Martin & Martin, 2016; Polumuri et al., 2012). In addition, mouse IL-33 has two alternative promoters. From these promoters, two full length IL-33 products are transcribed consisting of eight exons: IL- 33a or IL-33b. The production of either IL-33a or IL-33b is both tissue and cell dependent. For human IL-33, one shorter spliced version (lacking exon 3) exists (Polumuri et al., 2012; Talabot-Ayer et al., 2012). When expressed recombinantly, the short form of IL-33 is biologically active, although it is uncertain whether it is functional in vivo. IL-33 mRNA is degraded within hours after transcription. In addition, upon IFN-γ stimulation, STAT1 activates the large multifunctional protease 2 (LMP2) which decreases intracellular IL-33 by polyubiquitinated and subsequently degradation of protein IL-33. Hence, expression of IL-33 can be regulated on the mRNA and the protein level (N. T. Martin & Martin, 2016). IL-33 does not have a classical secretory leader sequence (Smith, 2010) and the process of IL-33 release is not yet fully understood.

However, in addition to necrosis and cell death, IL-33 can be released upon mechanical stress (Kakkar, Hei, Dobner, & Lee, 2012) and has been reported to be elevated in the serum in patients of many diseases (N. T. Martin & Martin, 2016; H. F. Zhang et al., 2012)).

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Figure 9. Regulation of IL-33 by production, release and processing. Human IL-33 is situated on chromosome 9 and can be transcribed into one long or one short form of IL- 33. Mouse IL-33 is present on chromosome 19 and can be transcribed into two transcripts (IL-33a or IL-33b). After IL-33 translation, protein IL-33 is translocated to the nucleus (A, B). Full length IL-33 can be processed by several proteases (calpain, esterases, cathepsin G and elastase) which increase IL-33 biological activity. However, after proteolytic cleavage with caspase-3, -7 or chymases IL-33 is rendered inactive. In addition, IL-33 is quickly oxidized outside the cells, which also inactivates its biological activity. Caspase-3 and -7 can inactivate IL-33 inside apoptotic cells or outside the cells (N. T. Martin & Martin, 2016). Used with permission.

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2.6.2 Extracellular IL-33 induced signaling and its receptor complex

If an intact cellular barrier is breached and cells destroyed, biologically active IL-33 is passively released; therefore IL-33 is defined as an alarmin, signaling cellular damage to the surrounding environment (Haraldsen, Balogh et al. 2009). Proteases derived from different cellular sources, as neutrophils and mast cells, process full-length IL-33 into N- terminally truncated forms (Human IL-33 95-270, 99-270, 109-270) that become up to 30 times more potent than the full-length form (Lefrancais & Cayrol, 2012; Lefrancais et al., 2014). Thus, IL-33 activity could potentially be drastically increased at the site of infection. However, the bioactivity is lost as soon as the IL-1 family core structure is cleaved. Thus, proteases like chymase (released by active mast cells) can both activate and inactivate IL-33 (Figure 9 B) (Lefrancais et al., 2014; N. T. Martin & Martin, 2016).

Apoptosis inactivates IL-33, likely by caspase-3 and -7 (N. T. Martin & Martin, 2016). By contrast, caspase-1 processing of IL-33 into a truncated form consisting of residue 112- 270 was first proposed to increase IL-33 bioactivity (Schmitz et al., 2005). Later, however, caspase-1 inactivates IL-33 by cleaving at position Asp178, not at the site initially proposed (Ser111) (Cayrol & Girard, 2009). In addition, inflammation-related caspases (caspase-1, -4 and -5) have been reported to not cleave in vitro translated IL-33 (Luthi et al., 2009). During necrotic conditions, in contrast to after apoptosis (Luthi et al., 2009), IL-33 is not enzymatically processed (Cayrol & Girard, 2009). However, processing of IL-33 is not necessary as full-length IL-33 (similar to IL-1 α) is biologically active N. T.

Martin & Martin, 2016) (figure 9 b). IL-33 signaling is mediated by binding to the IL-33 receptor ST2. This alters the conformation of ST2, facilitating interaction with IL-1RAcP and forming a transmembrane heterodimer complex. Furthermore, this structure has been resolved by nuclear magnetic resonance and small angle x-ray scattering (Lingel et al., 2009). The intracellular TIR heterodomains formed in this complex lead to the assembly of a signaling cascade consisting of MYD88, IRAK and TRAF6. This leads to activation of the NF-κB pathway (Schmitz et al., 2005) resembling IL-1β signaling. The NF-κB pathway is described in more detail in chapter 2.4.1 (Signaling in inflammation).

Relative to IL-33, IL-1β is a more potent activator of endothelial NF-κB signaling (Pollheimer et al., 2013). Quantitative phosphoproteomic analysis of IL-33 stimulated cells revealed altered phosphorylation of 672 proteins at 1050 sites (Pinto et al., 2015).

In addition, a microarray analysis of IL-33 stimulated HUVECs showed altered

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transcriptional levels of more than 300 genes (Pollheimer et al., 2013). A global proteomic high-resolution mass-spectrometry analysis of IL-33 stimulated endothelial cells revealed a distinct expression of proteins associated with an inflammatory response. In contrast, siRNA mediated depletion of IL-33 had no reproducible effect on the proteome. These observations suggest that IL-33 acts solely as a cytokine and not an intracellular gene regulating factor (Gautier et al., 2016). In compliance with this, IL-33 knockout mice develop normally, are healthy and fertile with now obvious phenotype in a pathogen-free environment (N. T. Martin & Martin, 2016). However, endogenous IL-33 contributes to development of allergic inflammation (airway inflammation and peripheral antigen-specific response in acute allergic lung inflammation (Louten et al., 2011)). In models of allograft rejection, tissue injury and pathology, administration of IL- 33 expands ILC2s and Tregs and protects from an exacerbated Th1 response. However, excess serum levels of IL-33 is also associates with autoimmunity and chronic pathology (Molofsky, Savage, & Locksley, 2015).

In addition, IL-33 signaling is required to establish an antiviral T-cell response upon LCMV infection of mice (Bonilla et al., 2012). These effects of IL-33 were shown in mice, however there are interspecies differences compared to human. Indeed, in humans, endothelial cells are a major reservoir for IL-33 (Kuchler et al., 2008), in contrast, constitutive IL-33 cannot be found in mouse endothelial cells (Pichery et al., 2012).

2.6.3 Intracellular Interleukin-33

The N-terminus of IL-33 contains a nuclear localization sequence responsible for directing IL-33 into the nucleus, heterochomatin and mitotic chromosomes (residues 1- 65) (Baekkevold et al., 2003; Carriere et al., 2007). Intracellular IL-33 can potentially regulate gene expression by different mechanisms. First, IL-33 binds to the acidic patch in histone H2A-H2B of heterochromatin, modulating a higher order chromatin structure, thus regulating transcription. Second, IL-33 associates with SUV39H1 and downregulates soluble ST2 (IL-1R4) and IL-6 expression. However, this is has only been published ones (to the best of my knowledge) and should ideally be validated by others. Lastly, IL-33 has also been reported to interact with NF-κB, thus reducing NF-κB DNA-binding activity (Ali et al., 2011; N. T. Martin & Martin, 2016). Nuclear localization of IL-33 is vital as disrupting the subcellular organization by altering the nuclear localization signal leads to

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non-resolving lethal inflammation (Bessa et al., 2014). These findings by Bessa et al (2014) show how crucial it is to control IL-33 localization, thus one function of sorting IL- 33 to the nucleus may be to store and prevent the detrimental effects of IL-33. In addition, after IL-33 is exposed to an oxidizing environment (as in the extracellular space) it is quickly oxidized, preventing IL-33-ST2 signaling (Cohen et al., 2015) thus contributing to an additional layer of functional IL-33 regulation.

2.6.4 Endothelial cells and IL-33

Endothelial IL-33 is present in most organs of the body. However, endothelial IL-33 is undetectable in the mouse (Pichery et al., 2012). Moreover, expression of human IL-33 is quite restricted to quiescent, non-proliferating endothelial (Kuchler et al., 2008;

Moussion, Ortega, & Girard, 2008) and rapidly downregulated upon angiogenic or acute pro-inflammatory activation (Kuchler et al., 2008).

2.6.5 The role of IL-33 in homeostasis and inflammation

The function of IL-33 is most frequently described as an activator of the type 2 immune response. However, recent studies extend IL-33 function to basal tissue regulation, organ-specific injury and repair as well as immunity to viruses, microbes and neoplasms.

Molofsky et al (2015) suggested a three step model of IL-33 action, including a homeostatic stage, an amplification stage and a conversion stage. At the homeostatic stage, constitutive pools of IL-33 sustain basal physiology by activating ST2 cells in a site- and tissue-specific manner. In addition to non-hematopoietic cells, including endothelial cells, epithelial cells and fibroblasts, innate lymphoid cells (ILC2s), T regulatory cells (Tregs) and mast cells are the primary tissue-resident cells constitutively expressing ST2, positioning these cells as initial targets for IL-33. The homeostatic role of IL-33 is best described in adipose tissue where it is highly expressed by endothelial cells and fibroblast-like reticular cells. Loss of IL-33 or ST2 in mice worsens obesity and insulin resistance (hallmarks of type 2 diabetes). In the amplification stage, tissue specific pools of IL-33 increase, as well as the expression of ST2 in an attempt to maintain homeostasis. This is usually in response to parasite infection or allergy which results in a manifestation of a Th2 response. A chronic amplification stage can result in pathogenic

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necrosis. In the conversion stage, tissue damage-induced inflammation leads to increased IL-33 responsiveness through induced expression of ST2 of inflammatory cells, including natural killer (NK), T helper type 1 (Th1) and CD8 T cells. In addition, regulatory and type 2 responses are also actively repressed by the Th1 cytokine IFN-γ. Thus, the response during the conversion phase facilitates clearance of pathogens but often with the side effect of tissue damage (Molofsky et al., 2015).

2.6.6 IL-33 and viral infections

IL-33 is essential for establishing protective immune responses against prototypic RNA and DNA viruses (lymphocytic choriomeningitis virus (LCMV), murine γ-herpes virus-68 (MHV-68) and vaccinia virus (VV)) in mice, activating both a CD8 cytotoxic response (Bonilla et al., 2012) and inducing transient ST2 expression in Th1 cells (Baumann et al., 2015). A genome wide cDNA expression analysis of the spleen in LCMV-infected mice revealed highly elevated levels of IFN-γ and IL-33. In addition, the IL-33 receptor ST2 was upregulated. Furthermore, the protective CD8 T cell response was disrupted in mice expressing a soluble decoy receptor for IL-33, in IL-33-deficient mice or in ST2-deficient mice. This indicates that the anti-viral effect of IL-33 was extracellular and receptor mediated. In contrast, the CD8 T cell response in ST2 knockout and WT mice was similar during infection with attenuated vaccinia virus, non-replicative adenovirus or non- replicative LCMV (Bonilla et al., 2012). This suggests that cellular lysis and release of IL- 33 are important for establishing the CD8 T cell response. This was confirmed by injecting IL-33 into infected mice, which augmented the CD8 response towards attenuated vaccinia virus or virus like particles (Bonilla et al., 2012).

During lung infection, IL-33 can be induced and released in response to an allergen (via ATP) and ATP stimulus (N. T. Martin & Martin, 2016). Influenza A virus infection of murine lungs drastically increases IL-33 expression (Le Goffic et al., 2011), and patients with chronic viral hepatitis have elevated levels of IL-33 in the blood (J. Wang et al., 2012). Furthermore, IL-33 is implicated in response to chronic viral infections caused by HIV (human immunodeficiency virus), HBV (Hepatit B virus) and HCV (Hepatit C virus), recently reviewed by Mehraj and Ponte et al (Mehraj et al., 2016). During adenoviral hepatitis, IL-33 modulates liver damage by activating ILC2s. In these mice, the liver and serum IL-33 expression increased six days post infection. Furthermore, injection of IL-33

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induced expression of Th1 cytokines IFN-γ and IL-2 but reduced the levels of the hepatoxic cytokine TNF-α. To confirm the effect of IL-33 on cytokine expression, intrahepatic lymphocytes (IHL) isolated from adenovirus-infected mice were cultured with or without IL-33. In CD8-, CD4- and CD11b-positive cells, IL-33 inhibits the expression of intracellular TNF-α. In addition, IL-33 induced Th1 cytokines (IFN-α and IL- 2), Th2 cytokines (IL-5 and IL-13) and type 2 chemokines (CCL17 and CCL22). Markers of viral hepatitis (councilman bodies and alanine aminotransferase (ALT)) were significantly reduced in the IL-33 treated mice, suggesting that IL-33 has a protective role in adenovirus induced hepatitis. Furthermore, IL-33 treatment significantly reduced monocytes and NK cells but increased Tregs among the splenic and intrahepatic lymphocytes. In conclusion, IL-33 stimulates anti-inflammatory ILC2s and Tregs, and reduces the level of the hepatocytic cytokine TNF-α, which consequently have a protective role against adenovirus-induced hepatits (Liang et al., 2013).

2.7 The DNA damage response (DDR)

Damage to the DNA can be caused by oxidative stress (Zhan, Suzuki, Aizawa, Miyagawa,

& Nagai, 2010), irradiation and chemicals (Pamment et al., 2002) leading to double or single stranded breaks (DSB/SSB) to the DNA.

There are two types of double stranded break repair: The first one, homologous recombination repair (HRR), using homologous DNA template to repair DNA breaks in an error free manner, which occurs during the S or G2 phase. The second repair mechanism is non-homologous end joining repair (NHEJ) that directly rejoins double stranded breaks, which is active throughout the cell cycle and is prone to DNA error (Ciccia &

Elledge, 2010).

DNA breaks are recognized by DNA-dependent protein kinase catalytic subunits (DNA- PKcs)(Carter, Vancurova, Sun, Lou, & DeLeon, 1990; Lees-Miller, Chen, & Anderson, 1990) and Ku70/80 (Dvir, Peterson, Knuth, Lu, & Dynan, 1992; Gottlieb & Jackson, 1993)), ATM and the MRN protein complex (Lee & Paull, 2004, 2005; Uziel et al., 2003) or ATM-RAD3 Related (ATR) and replication protein A (RPA) (Cimprich, Shin, Keith, &

Schreiber, 1996; Cliby et al., 1998; Zou & Elledge, 2003). While DNA-PKcs and Ku sensors

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are mainly involved in non-homologous end joining repair, ATM and MRN mediates signaling in response to homologous recombination repair (Lee & Paull, 2005). ATR and RPA are recruited to sites of DNA damage binding to ssDNA with the help of ATR- interacting protein (Cortez, Guntuku, Qin, & Elledge, 2001; Zou & Elledge, 2003).

ATM was discovered in 1995 as the gene mutation leading to defects (predisposition to cancer and radio sensitivity) causing the human autosomal recessive syndrome ataxia telangiectasia (Lavin, 2008; Savitsky et al., 1995). The ATM signaling pathway is activated in response to non-replicative Ad5 and E4 deleted Ad5, where DNA damage response components are localized to viral replication centers (Carson et al., 2003). The MRE11- RAD50-NBS1 complex (MRN), required for ATM recruitment, is involved in concatemer formation of adenovirus (ligation of Ad5 genomes) (Stracker et al., 2002). The adenoviral proteins E1-B55k and E4ORF3 target the DNA damage response component MRE11 for proteaosomal degradation, which is critical for viral genome replication (Carson et al., 2003; Shah & O'Shea, 2015). Furthermore, this inhibits the DNA damage response in Hela cells, U2OS cells (Carson et al., 2003) and in human small airway epithelial cells (SAECs)(Shah & O'Shea, 2015). Another form of DNA damage response can be activated by oxidative stress caused by hydrogen peroxide or viral endosome rupture leading to phosphorylation of ATM and downstream phosphorylation of AKT, p53 and induction of P21 (McGuire, Barlan, Griffin, & Wiethoff, 2011; Zhan et al., 2010). This induces endothelial cell senescence through ATM activation (Zhan et al., 2010). In addition, hydrogen peroxide-induced oxidative stress has been shown to induce NOTCH1 signaling, connecting DNA damage response to NOTCH1 (Boopathy, Pendergrass, Che, Yoon, & Davis, 2013).

2.7.1 The DNA damage response and activation of innate immunity

DNA damage does not only result in cell cycle arrest (as described above) but can lead to activation of the immune system. Treating cells with etoposide (causing double stranded DNA breaks) activates an IFN response by inducing expression IRF-1 and -7 (Brzostek- Racine, Gordon, Van Scoy, & Reich, 2011). Ku70 functions as a cytosolic DNA sensor leading to IFNL1 (type 3 IFN response) via IRF-1 and IRF-7 by binding to PRD1 and ISRE domains (Xing Zhang et al., 2011). In addition, DNA-PKcs DNA sensing activates IRF-3, TBK1 and STING, leading to expression of IFN-β independent on NF-κB signaling