Peripheral Regulation of Pain and Itch

Home , CPA3, Endothelin 3, Endothelin A receptor, Endothelin B receptor, Endothelin receptor, Exopeptidase inhibitor

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1596

ELÍN INGIBJÖRG MAGNÚSDÓTTIR

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-513-0746-6 UPPSALA urn:nbn:se:uu:diva-392709 2019 Dissertation presented at Uppsala University to be publicly examined in A1:107a, BMC, Husargatan 3, Uppsala, Friday, 25 October 2019 at 13:00 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Professor emeritus George H. Caughey (University of California, San Francisco).

Abstract Magnúsdóttir, E. I. 2019. Peripheral Regulation of Pain and Itch. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1596. 71 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0746-6.

Pain and itch are diverse sensory modalities, transmitted by the somatosensory nervous system. Stimuli such as heat, cold, mechanical pain and itch can be transmitted by different neuronal populations, which show considerable overlap with regards to sensory activation. Moreover, the immune and nervous systems can be involved in extensive crosstalk in the periphery when reacting to these stimuli. With recent advances in genetic engineering, we now have the possibility to study the contribution of distinct neuron types, neurotransmitters and other mediators in vivo by using gene knock-out mice. The neuropeptide calcitonin gene-related peptide (CGRP) and the ion channel transient receptor potential cation channel subfamily V member 1 (TRPV1) have both been implicated in pain and itch transmission. In Paper I, the Cre- LoxP system was used to specifically remove CGRPα from the primary afferent population that expresses TRPV1. CGRPα-mCherrylx/lx;Trpv1-Cre mice had attenuated responses to visceral pain induced by acid, while mechanosensitivity of the colon and somatic pain sensation remained unaffected. Mast cell proteases (MCPs) are stored in high quantities within mast cell (MC) granules and have been linked to both protective and pro-inflammatory properties, but little is known about their exact roles in vivo. In Papers II, IV and V, we used knock-out mice to investigate the contribution of MCs and their MCPs (the chymase mMCP4, tryptase mMCP6 and carboxypeptidase CPA3) in pain resulting from tissue injury, inflammation-induced heat hypersensitivity and different types of itch. Surprisingly, we found that neither MCPs nor MCs were essential for the pain behavior tested (Paper II). Our data indicate that mMCP6 and CPA3 have a protective role in scratching behavior induced by the peptide endothelin-1 (ET-1; Paper IV) and in scratching induced by the MC degranulator compound 48/80 (Paper V), but no differences were observed with the other pruritogens histamine, chloroquine or SLIGRL. In Paper III, we saw that a novel single-stranded oligonucleotide (ssON) attenuated compound 48-induced scratching in BALB/c mice by blocking MC degranulation. ssON could also block degranulation in human MC in vitro and we determined that this was due to ssON interfering with Mas-related G protein-coupled receptor X2 (MRGPRX2), a receptor involved in non-allergic MC degranulation. By better understanding the contribution of individual components of the nervous and immune systems in pain and itch, we hopefully increase the possibilities of developing better treatments for burdensome pain- and itch-related disorders in the future.

Keywords: Pain, itch, CGRP, TRPV1, mast cell, mast cell protease, mMCP4, mMCP6, CPA3, endothelin-1, compound 48/80, MRGPRX2, single-stranded oligonucleotide

Elín Ingibjörg Magnúsdóttir, Department of Neuroscience, Box 593, Uppsala University, SE-75124 Uppsala, Sweden.

ISSN 1651-6206 ISBN 978-91-513-0746-6 urn:nbn:se:uu:diva-392709 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-392709)

Til Steinars, Ylfu og Aríu

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Spencer, N. J.*, Magnúsdóttir, E. I.*, Jakobsson, J. E. T., Kestell, G., Chen, B. N., Morris, D., Brookes, S. J., Lagerström, M. C. (2018). CGRPα within the Trpv1-Cre population contributes to visceral nociception. American Journal of Physiology – Gastro- intestinal and Liver Physiology, 314(2), G188-G200. *shared first authorship

II Magnúsdóttir, E. I., Grujic, M., Roers, A., Hartmann, K., Pejler G., Lagerström, M. C. (2018). Mouse mast cells and mast cell proteases do not play a significant role in acute tissue injury pain induced by formalin. Molecular Pain, 14, 1-11.

III Dondalska, A., Rönnberg, E., Ma, H., Pålsson, S., Magnúsdóttir, E. I., Gao, T., Adam, L., Lerner, E. A., Nilsson, G., Lagerström, M. C., Spetz, A. Amelioration of Mas-related G protein-coupled receptor X2-mediated itch and reduced mast cell degranulation by a single-stranded oligonucleotide. Manuscript.

IV Magnúsdóttir, E. I., Grujic, M., Bergman, J., Pejler, G., Lager- ström, M. C. Mouse mast cell tryptase and carboxypeptidase A3 play a protective role in itch induced by endothelin-1. Manu- script.

V Magnúsdóttir, E. I., Grujic, M., Pejler, G., Lagerström, M. C. Compound 48/80-induced itch is attenuated by mouse mast cell tryptase and carboxypeptidase A3. Manuscript.

Reprints were made with permission from the respective publishers.

Additional publications

Rogoz, K., Aresh, B., Freitag, F. B., Pettersson, H., Magnúsdóttir, E. I., Larsson Ingwall, L., Haddadi Andersen, H., Franck, M. C., Nagaraja, C., Kullander, K., Lagerström, M. C. (2016). Identification of a neuronal receptor controlling anaphylaxis. Cell Reports, 14(2), 370-379.

Contents

Introduction ...... 11 Background ...... 13 The sensory pathways of pain and itch ...... 13 Ion channels and receptors in nociception ...... 14 Nociception in the gut ...... 15 Pruriception ...... 16 Histaminergic itch ...... 17 Non-histaminergic itch ...... 18 A tale of two peptides involved in pain and itch ...... 21 Calcitonin gene-related peptide ...... 21 Endothelin-1 ...... 22 The effects of CGRP and ET-1 on immune cells ...... 24 Mast cells...... 27 Mast cell receptor activation ...... 27 Mast cell proteases ...... 29 Chymase ...... 30 Tryptase ...... 31 Carboxypeptidase ...... 31 Monoclonal antibodies against pain and itch ...... 34 Aims ...... 37 Results and discussion ...... 38 Paper I ...... 38 Paper II ...... 40 Paper III ...... 42 Paper IV ...... 43 Paper V ...... 45 Summary and future perspectives ...... 48 Acknowledgments...... 50 References ...... 52

Abbreviations

5HTR 5-hydroxytriptamine (serotonin) receptor AD Atopic dermatitis ATP Adenosine triphosphate BAM 8-22 Bovine adrenal medulla peptide 8-22 CBMC Cord blood-derived mast cell CGRP Calcitonin gene-related peptide CPA3 Carboxypeptidase A3 CRLR Calcitonin receptor-like receptor CTMC Connective tissue mast cell DRG Dorsal root ganglia ET-1, -2, -3 Endothelin-1, -2, -3

ETR, ETAR, ETBR Endothelin receptor, A, B GPCR G protein-coupled receptor H1, H2, H3, H4 Histamine receptor 1, 2, 3, 4 HEK293 Human embryonic kidney cell line 293 HMGB1 High mobility group protein B1 I.p. Intraperitoneal IB4 Isolectin B4 IFNγ Interferon gamma IgE, IgG Immunoglobulin E, G IL Interleukin mAb Monoclonal antibody MC, MMC Mast cell, mucosal mast cell mMCP Mouse mast cell protease moDC Monocyte-derived dendritic cell Mrgpr Mas-related G protein-coupled receptor MrgprA3 Mrgpr member A3

Mrgprb2 Mrgpr member B2 MrgprC11 Mrgpr member C11 MrgprD Mrgpr member D MRGPRX2 Mrgpr member X2

NaV Voltage-gated sodium channel NGF/β Nerve growth factor/β NK1R Neurokinin-1 receptor Nppb Natriuretic polypeptide B PAR2 Protease-activated receptor 2 PLA2 Phospholipase A2 PLCβ3 Phospholipase Cβ3 Poly I:C Polyinosinic:polycytidylic acid qPCR Quantitative polymerase chain reaction RAMP1 Receptor activity-modifying protein 1 SLIGRL Ser-Leu-Ile-Gly-Arg-Leu SP Substance P ssON Single-stranded oligonucleotide TG Trigeminal ganglia TLR Toll-like receptor TNF/α Tumor necrosis factor /α TrkA Tropomyosin receptor kinase A TRP Transient receptor potential TRPA1 TRP cation channel subfamily A member 1 TRPM8 TRP cation channel subfamily M member 8 TRPV1 TRP cation channel subfamily V member 1 TRPV4 TRP cation channel subfamily V member 4 TSLP/R Thymic stromal lymphopoietin/receptor VGLUT2 Vesicular glutamate transporter 2 VIP Vasoactive intestinal peptide VPAC2 VIP receptor type 2

Introduction

“Everyone is down on pain, because they forget something important about it: Pain is for the living. Only the dead don’t feel it.

Pain is a part of life. Sometimes it’s a big part, and sometimes it isn't, but either way, it's a part of the big puzzle, the deep music, the great game. Pain does two things: It teaches you, tells you that you’re alive. Then it passes away and leaves you changed. It leaves you wiser, sometimes. Sometimes it leaves you stronger. Either way, pain leaves its mark, and everything important that will ever happen to you in life is going to involve it in one degree or another.” ― Jim Butcher, White Night

Pain is a phenomenon that we mostly associate with negative emotions. It can refer to the stabbing physical pain of stubbing your toe on the living room couch, the emotional pain of losing a loved one, or the chronic debilitating pain of arthritis. We learn early on to avoid pain, and this instinct often keeps us out of harm’s way. Therefore, pain generally serves an important purpose, however much we may dislike the actual sensation. When we experience acute physical pain, it is usually an indicator of the presence of a harmful stimulus, such as a hot stove or a needle prick. In chronic pain however, the underlying physical conditions are more complicated. Triggers such as injury or disease can cause long-lasting systematic changes, for example nerve damage (neuropathic pain) or persistent inflammatory states. A sensation of that kind no longer serves its purpose, and instead causes suffering, worsens the quality of life and even results in debilitation for the afflicted individual. Chronic pain is a major health care problem; for instance, it has been estimated that around one in every five individuals in Europe suffers from chronic pain, and 40% of those patients go without adequate pain management (Breivik et al. 2006). Itch is another concept with mostly negative connotations; besides the actual physical sensation of itch, we use the word itself to describe irritation and restlessness. Acute physical itch is usually the indicator of a stimulus on the skin, such as a crawling insect or an irritating substance, which can be promptly removed by scratching or touching the affected area. For most people, itch is not a particularly serious or dramatic problem to consider, even though we are all familiar with the irritating itch that can bother us for a few days after a mosquito bite. But what if that sensation persisted for weeks, or even longer? In chronic itch, just as in chronic pain, a long-lasting underlying

11 condition is the source of the sensation. Chronic itch can accompany skin diseases like eczema and atopic dermatitis, systemic disorders such as chronic kidney disease and liver dysfunction, or nervous system disorders, such as multiple sclerosis (Ikoma et al. 2006). Around one in four people will experience chronic itch (lasting for at least six weeks) at some point in their life (Ständer et al. 2019). In certain cases, such as in prurigo, a skin disease characterized by intense pruritus and accompanying lesions, the impact on the quality of life is especially severe, and one study reported suicidal ideation in one out of five patients (Brenaut et al. 2019). Scratching in response to chronic itch is usually futile and even harmful to the skin, and treatment options are commonly limited and need to be tailored to the underlying etiology and understanding of the respective condition in order to be effective (Stull, Lavery, and Yosipovitch 2016). Pain and itch may seem like very different sensations but upon further in- spection we discover that they have several important aspects in common: they are unpleasant sensations that may help protect us from the environment, they are transmitted via similar neuronal mechanisms, in chronic conditions they can severely affect the patients’ quality of life and there is a dire need for improved treatment options. In order to develop new treatments, we need to increase our understanding of the underlying mechanisms behind pain and itch, and in this thesis I will discuss several of the factors that regulate acute pain and itch in the periphery.

12 Background

The sensory pathways of pain and itch Pain and itch are detected by somatosensory neurons. The cell bodies of these neurons are gathered in ganglia that either lie alongside the spinal cord, called dorsal root ganglia (DRG), or are situated at the skull base, referred to as trigeminal ganglia (TG). The sensory nerve endings of TG neurons cover the head and face, and the DRG neurons get input from the rest of the body. In addition, pain from the internal organs of the body can also be transmitted via the vagus nerve (Gebhart and Bielefeldt 2016). The DRG and TG neurons are pseudo-unipolar, i.e. they have a single axon that branches out and projects both to the periphery and to the dorsal horn of the spinal cord (Basbaum et al. 2009). In the spinal cord, sensory information is integrated with the help of interneurons and finally transmitted to the thalamus and brainstem; the major pathways for that are the spinothalamic and spinoparabrachial tracts (Braz et al. 2014). Further processing and interpretation of the sensory information then takes place in the brain. Traditionally, somatosensory neurons have been categorized into four general fiber types, also referred to as primary afferents: Aα, Aβ, Aδ and C fibers. Pain and itch are detected by subpopulations of the primary afferents, or nociceptors and pruriceptors, respectively (Ikoma et al. 2006; Basbaum et al. 2009). Correspondingly, pain and itch sensation can be referred to as nociception and pruriception. Nociceptors are generally divided into two major classes, myelinated Aδ afferents and unmyelinated C fibers. The Aδ afferents respond to temperature, chemical irritants and force, and mediate acute and “fast” or “first” pain (Basbaum et al. 2009). The C fibers can respond to tissue damage, chemical irritants (including pruritogens, i.e. itch-inducing substances), temperature and mechanical force, and transmit “slower” or “second” pain that often is poorly localized (Schmelz et al. 1997; Basbaum et al. 2009). Low-threshold mechanoreceptor C fibers are activated by light touch, which is perceived as pleasant in humans (Löken et al. 2009) and can have a role in attenuating pain (Habig et al. 2017). C fibers are estimated to make up around 50% of all somatosensory neurons and they are considered the major neuronal population responsible for sensing pain and itch in humans and other mammals (Schmelz et al. 1997; Ikoma et al. 2006; Basbaum et al. 2009; Sneddon 2018). C fibers are classically divided

13 into two categories based on which neurotransmitters are present, they are referred to as peptidergic where neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P (SP) can be found, and non-peptidergic, where the histological marker isolectin B4 (IB4) binds (Averill et al. 1995). Recently, advances in single-cell RNA sequencing have made it possible to characterize the neurons even further, utilizing transcriptomics to organize them in clusters based on their function and gene expression profile (Gatto et al. 2019). To put the results of such analysis in context we first need to take a closer look at the ion channels and receptors that are important in pain and itch sensation.

Ion channels and receptors in nociception Sensory neuronal populations are commonly categorized based on the type of receptors and ion channels they express. The transient receptor potential (TRP) ion channel family consists of around 30 members (Nilius et al. 2007), some of which have been associated with sensory signaling. The TRP cation channel subfamily V member 1 (TRPV1) is a ligand-gated ion channel found in nociceptive afferents (Caterina et al. 1997) and is present in nearly all unmyelinated peptidergic DRG neurons, but is mostly absent from IB4-binding non-peptidergic C fibers (Cavanaugh et al. 2011). TRPV1 can be activated by heat over 43°C, capsaicin (the active ingredient that gives chili peppers their pungency) and low pH (Caterina et al. 1997), as well as anandamide (a cannabinoid receptor agonist; Zygmunt et al. 1999). In knock-out models in mice, it has been found that those lacking TRPV1 have lower behavioral responses to noxious heat and reduced inflammation-induced thermal hyperalgesia (Caterina et al. 2000; Davis et al. 2000) but respond normally to noxious mechanical stimuli (Caterina et al. 2000). Another member of the TRP family, the cation channel subfamily A member 1 (TRPA1), is expressed in a subpopulation of TRPV1-positive neurons (Story et al. 2003; Mishra and Hoon 2010) and can be activated by various chemical irritants, such as isothiocyanates (from mustard and horseradish), tetrahydrocannabinol (Jordt et al. 2004) and formalin (McNamara et al. 2007). TRP cation channel subfamily M member 8 (TRPM8) is activated by low temperatures (8-26°C) and menthol (McKemy, Neuhausser, and Julius 2002), but not by noxious cold (-10°C-0°C) (Bautista et al. 2007). TRPM8 shows almost complete overlap with TRPV1 neurons during development but little overlap in adults (Mishra et al. 2011). The tropomyosin receptor kinase A (TrkA) is a high-affinity receptor for nerve growth factor (NGF) and its activation in peptidergic afferents is of importance in nociception (Snider and McMahon 1998). When activated with NGF, TrkA increases the expression of TRVP1 channels, thereby sensitizing

14 the neuron to stimuli such as noxious heat (Zhang, Huang, and McNaughton 2005). Therefore, NGF injections in mice have become a well established model to study heat hypersensitivity, and this method is used in Papers I and II. Voltage-gated sodium channels (NaV) that play an important role in pain transmission have been identified, such as NaV1.7 and NaV1.8 (Akopian et al. 1999; Nassar et al. 2004). NaV channels are only expressed in peripheral nociceptors (mostly in C fibers); NaV1.7 is important for mediating inflammatory pain (Drenth et al. 2001; Nassar et al. 2004) and NaV1.8 plays a role in sensing noxious mechanical stimuli as well as inflammatory pain and noxious cold (Akopian et al. 1999; Abrahamsen et al. 2008; Lagerström et al. 2011). Dif- ferent point mutations in the NaV1.7 gene (SCN9A) have been reported in humans, they can result in lowered pain thresholds and episodes of spontaneous, burning pain (primary erythermalgia; Yang et al. 2004), and another mutation presents as insensitivity to pain (Cox et al. 2006; Yuan et al. 2011). The non-peptidergic C fiber population in DRGs expresses receptors of the Mas-related G protein-coupled receptor (Mrgpr) family (Dong et al. 2001). These include Mrgpr member D (MrgprD), which has been found to be involved in sensing noxious mechanical stimuli and β-alanine-induced itch (Cavanaugh et al. 2009; Q. Liu et al. 2012) and Mrgpr member A3 (MrgprA3), which mediates chloroquine-induced itch (L. Han et al. 2013). When the above information is integrated and combined with results from single-cell RNA sequencing, a few distinct types of sensory cells have been defined in mice (Gatto et al. 2019). For instance, a cluster of peptidergic cells has been categorized that express the cool sensor TRPM8 and SP, but not CGRP. C fiber nociceptors that express CGRP have been identified as a special cluster, as well as CGRP-positive Aδ nociceptors. In the non-peptidergic cells, there is a type that expresses MrgprD and is important in sensing mechanical pain, and two types that are specific for itch, characterized by their expression of MrgprA3 and the transmitter natriuretic polypeptide B (Nppb), respectively (Li et al. 2016; Usoskin et al. 2015; Zeisel et al. 2018; summarized in Gatto et al. 2019).

Nociception in the gut Pain arising from the viscera, i.e. the internal organs in the chest, abdomen and pelvis, is not as well understood as pain experienced from other parts of the body. Research has historically been limited by the fact that the skin is easily accessible to test various physical stimuli, while the viscera are not. Moreover, the visceral sensory innervation is complex but sparse compared with the skin, which makes visceral pain often diffuse and difficult to localize (Gebhart and Bielefeldt 2016). The viscera are innervated by the vagus nerve (Cervero 1994) as well as by spinal afferents (DRGs) (Spencer, Kyloh, and Duffield 2014).

15 In the gut wall, five major types of structural sensory endings have been described, mostly they are activated by factors important for digestion and gastrointestinal motility, such as distension, pH and mediators released from enteroendocrine cells (Brookes et al. 2013). However, potential nociceptors were also found in both spinal and vagal afferents, that frequently were TRPV1-positive and expressed CGRP (Brookes et al. 2013). TRP cation channel subfamily V member 4 (TRPV4) and TRPA1 channels are also commonly found in the gut, where TRPV4 can respond to osmotic swelling and mechanical distortion (Nilius et al. 2004; Brierley et al. 2008) and TRPA1 is mecha- nosensitive and interacts with TRPV1 (Nilius, Prenen, and Owsianik 2011). Although somatic sensation is the main focus of this thesis, in Paper I we also take a look at visceral nociception, where we investigate the role of CGRPα within the TRPV1 population.

Pruriception Pain and itch are related sensations and affect each other in various ways. For example, pain inhibits itch, as experienced when we scratch, and it has been shown that this is due to scratch-induced inhibition of spinothalamic tract neurons (Davidson et al. 2009). Opioid analgesics can induce itch (X.-Y. Liu et al. 2011), and algogens (pain-inducing substances) are also capable of that, for instance when capsaicin is topically applied (Sikand et al. 2009). Under certain conditions pruritogens, such as histamine and spicules from the plant cowhage (Mucuna pruriens var. pruriens), can induce pain (LaMotte et al. 2009; Sikand et al. 2009). Another example is the peptide endothelin-1 (ET- 1), which is generally algogenic when exogenously administered (Dahlof et al. 1990) but can also induce pruritus when injected intradermally in low doses, and produces itch that is described as “burning”, i.e. pruritus with painful qualities (Ferreira, Romitelli, and de Nucci 1989; Namer et al. 2008). An interesting distinction between pain and itch sensation is that pain can be felt from almost every part of the body, while itch, whether it is due to a skin condition or a systematic disorder, is only sensed on the surface of the body, such as in skin and external mucosa (Greaves and Khalifa 2004; LaMotte, Dong, and Ringkamp 2014). It is still debated whether there are distinct subsets of nociceptors that mainly sense itch but not pain, or if the same neurons are involved in both sensations but activated at different thresholds or patterns of stimulation (McMahon and Koltzenburg 1992; L. Han et al. 2013; Braz et al. 2014). An- other complementary hypothesis is that pain activates central inhibitory mechanisms that can block itch pathways (Lagerström et al. 2010; Ross et al. 2010). The theory of population coding integrates these hypotheses in a model that proposes that sensory-labeled lines of afferents exist, and that they are inter- connected to other populations by interneurons and have the ability to activate

16 or inhibit each other (Ma 2012). Further, multiple polymodal afferents can be activated by a single stimulus and crosstalk between them is required to generate one dominant sensation (Ma 2012). Chemical itch is the term used to describe it when itch-sensing, unmyelinated C fiber afferents (pruriceptors) in the skin are activated by various different pruritogens, which in broad terms are classified as either histaminergic or non-histaminergic (Schmelz et al. 1997). These afferents generally express the TRPV1 ion channel (Imamachi et al. 2009; Liu et al. 2009; Mishra and Hoon 2013). Mechanical (or non-chemical) itch can be instigated via light touch on hairy skin, which activates afferents that are myelinated and touch-related (low- threshold mechanoreceptors), and it has been suggested that the ascending transmission pathway for mechanical itch is distinct from chemical itch (Bourane et al. 2015). This form of light, localized stimulation could be viewed as light tickling, which does instigate a scratching response, while the rougher form of tickling, rhythmic pressure that moves around the body, does not (Mintz 1967). Other categories of itch have been defined as well, such as neuropathic itch instigated by injury or disease of the somatosensory nervous systems, neurogenic itch resulting from neuronal transmission abnormalities, and psychogenic itch, which can accompany certain psychiatric disorders such as parasitophobia (Greaves and Khalifa 2004). The above examples of itch arising from the nervous system would be considered chronic pruritus. In further discussions on itch in this thesis we will focus mainly on the acute chemical variety.

Histaminergic itch The most extensively studied pruritogen, histamine, is a biogenic amine that is released from mast cells, basophils and keratinocytes when they are activated (Falcone, Knol, and Gibbs 2011; Inami et al. 2013), for example in allergic reactions when an immunoglobulin E (IgE) antibody crosslinks the FcεRI receptor on the cell surface (Jouvin et al. 1994). Histamine induces itch by binding to and activating histamine receptors on TRPV1-expressing sensory neurons (Shim et al. 2007; Imamachi et al. 2009). The histamine receptors are G protein-coupled receptors (GPCRs), of which four subtypes have been identified, histamine receptors 1, 2, 3 and 4 (H1, H2, H3, H4; Bell, McQueen, and Rees 2004). Acute histamine-induced itch in humans has been attributed to H1 (Davies and Greaves 1980) while H1, H4 and possibly H3 mediate itch in mice (Bell et al. 2004). Binding of histamine causes conformational changes of the receptor, which initiate intra- cellular secondary messenger pathways, either through phospholipase Cβ3 (PLCβ3; Han, Mancino, and Simon 2006; Imamachi et al. 2009) or phospholipase A2 (PLA2; Shim et al. 2007). This results in opening of the TRPV1 ion

17 channels, and subsequent influx of sodium, potassium and calcium ions depo- larizes the neuron. The depolarization opens NaV channels and further influx of sodium generates an action potential, propagating the itch signal further to the dorsal horn of the spinal cord (Imamachi et al. 2009). See Figure 1 for a simplified summary of the pathway described above. The other main pruritogen that is generally classified as histaminergic is compound 48/80, which promotes mast cell degranulation and subsequent histamine release (Barrett, Ali, and Pearce 1985). However, compound 48/80 also induces the release of other pruritogens from mast cells, such as tryptase and serotonin (Meixiong et al. 2019). Urticaria (hives) is a skin rash predominantly caused by histamine release from skin mast cells (Radonjic-Hoesli et al. 2018). It is characterized by patches of red, itchy and raised bumps (wheals), and is most commonly due to an IgE-mediated allergic reaction. Triggers also include pseudo-allergic reactions, and physical stimuli such as heat and cold (Radonjic-Hoesli et al. 2018). In most cases, the rash disappears within 24 hours but if the reaction reoccurs repeatedly for a longer period than six weeks, the urticaria is termed chronic. The first-line treatment for both acute and chronic urticaria is H1 antagonists but for non-responders in chronic urticaria, a monoclonal antibody (mAb) against IgE, omalizumab, has been shown to be effective (Kolkhir et al. 2019).

Non-histaminergic itch The majority of itch-inducing substances are non-histaminergic, and chronic itch, with the exception of urticaria, is generally of such origin. Therefore, classical histamine receptor antagonists are usually not effective in treating those conditions (Stull et al. 2016) and other receptors need to be considered instead. The Mrgpr receptors are a prominent target of non-histaminergic pruritogens. In mice, they include MrgprA3, which is activated by the anti-malaria drug chloroquine (Liu et al. 2009), Mrgpr member C11 (MrgprC11), which is activated by the endogenous opioid bovine adrenal medulla peptide 8-22 (BAM 8-22) (Liu et al. 2009) and the synthetic peptide Ser-Leu-Ile-Gly-Arg- Leu (SLIGRL) (Q. Liu et al. 2011), and MrgprD, which responds to β-alanine, an endogenous amino acid (Q. Liu et al. 2012). Another example of a non-histaminergic itch receptor is the protease-activated receptor 2 (PAR2), it is a GPCR activated by the endogenous enzymes mast cell tryptase and pancreatic trypsin (Corvera et al. 1997; Cottrell et al. 2004). The enzymes cleave a part of the receptor, creating a tethered ligand, which binds to the receptor and activates it (Ramachandran and Hollenberg 2009). SLIGRL mimics the sequence of the tethered ligand and thus activates PAR2 and MrgprC11 (Q. Liu et al. 2011).

18 The endothelin receptors A (ETAR) and B (ETBR) are GPCRs that are widely expressed in the body, including sensory neurons (Davenport et al. 2016). They appear to have opposite functions: ET-1-mediated activation of ETAR elicits scratching, while the same response occurs when ETBR is inhibited (McQueen, Noble, and Bond 2007). Fourteen different serotonin receptors (5-hydroxytriptamine receptors; 5- HTRs) have been described (of which 13 are GPCRs), they are widely expressed as serotonin has a multitude of different physiological functions, such as modulation of appetite, memory, mood, peristalsis in the gastrointestinal tract and vasoconstriction (McCorvy and Roth 2015). Serotonin is also a potent pruritogen and a few of the 5HTRs have been identified as important receptors for this function, for example 5HT1R, 5HT2R (Kim et al. 2008; Imamachi et al. 2009) and 5HT7R (Morita et al. 2015). Toll-like receptor 3 (TLR3) is an example of an itch receptor that is not a GPCR, but rather a pattern recognition receptor (Takeda, Kaisho, and Akira 2003). TLRs are typically expressed on immune and glial cells, where they play an important part in innate immunity by recognizing molecular motifs associated with pathogens or injured tissue (Takeda et al. 2003), but TLR3 is also expressed in the peripheral and central nervous system of mice (Cameron et al. 2007). It recognizes double-stranded RNA and is also activated by the synthetic ligand polyinosinic:polycytidylic acid (poly I:C), which induces scratching in wild-type mice when injected, but not in Tlr3-/- mice (T. Liu et al. 2012). A novel single-stranded nucleotide (ssON) has been shown to inter- fere with TLR signaling (Järver et al. 2018) and in Paper III we investigate its effects on mast cell degranulation and itch. The thymic stromal lymphopoietin receptor (TSLPR) is a heterodimer, which contains both the IL-7 receptor alpha chain and a TSLP-specific receptor chain (Pandey et al. 2000). It is activated by the epithelial cell-derived cytokine TSLP and this type of activation has been reported in various immune cells, such as basophils, mast cells, T cells and dendritic cells (Ziegler et al. 2013). Wilson and colleagues (2013) showed that TSLPRs are also expressed in mouse sensory neurons, and exogenous TSLP administration resulted in a robust scratching behavior, which was mediated via TSLPR together with TRPA1. TSLP is highly expressed in the skin of atopic dermatitis (AD) patients and is considered one of the factors that contribute to the development of the disease by stimulating cytokine secretion from immune cells (Jariwala et al. 2011) and by inducing pruritus directly by acting on sensory nerves (Wilson et al. 2013). Another cytokine with a similar function and that is strongly implicated in AD as well, is the T-cell derived IL-31 (Grimstad et al. 2009). It acts on the IL-31 receptor A, which is expressed on epithelial cells and keratinocytes (Dillon et al. 2004), and on sensory afferents (Sonkoly et al. 2006). IL-31 is upregulated in the skin of patients with pruritic inflammatory skin diseases such as AD, and especially high levels were observed in patients with prurigo

19 nodularis, a condition characterized by intense itching and lesions (Sonkoly et al. 2006). The examples listed here do not represent a complete list of known pruritogens and their receptors, but they demonstrate the diversity of the different pathways and molecules that are capable of inducing pruritus. For a schematic overview of several of the different cell types, receptors and ion channels that are involved in pruriception, see Figure 1. Variables such as skin status, temperature, underlying disease and species- specific differences can also play an important role in the function of the pruritogens. Thus, it becomes clear that effective treatment against itch, especially of the chronic variety, needs to be tailored to each patient and based on understanding the respective disorder and the underlying mechanisms.

Figure 1. Examples of cell types, receptors and ion channels involved in pruriception. The axons (primary afferents) of DRG sensory neurons extend into the skin. Pruritogens, either exogenous or endogenous (from keratinocytes, immune cells or neurons) can activate distinct subsets of these afferents. Histamine activates H1, which leads to the opening of TRPV1 channels (red). The pruritogens BAM8-22, chloroquine and TSLP activate the MrgprC11, MrgprA3 and TSLPR receptors, respectively, which leads to the opening of TRPA1 channels (orange). Influx of ions through TRPV1 and TRPA1 leads to the depolarization of the neuron, which opens NaV channels (black). Further influx of sodium ions through NaV triggers action potential firing and propagation of the itch signal further to the central nervous system. Complicated crosstalk can take place in the skin, where skin cells, immune cells and neurons can affect each other using various signaling molecules and mediators. Ab- breviations: DRG, dorsal root ganglia; H1, histamine 1 receptor; TRPV1/4, transient receptor potential cation channel subfamily V member 1/4; MrgprC11/A3, Mas-related G protein-coupled receptor member C11/A3; TSLP/TSLPR, thymic stromal lymphopoietin/receptor; NaV, voltage-gated sodium channel. Figure from Bautista, Wilson and Hoon (2014). Reprinted with permission from the publisher.

20 A tale of two peptides involved in pain and itch A variety of different mediators in the periphery can participate in inflammatory reactions and affect pain and itch transmission, many of which have been mentioned above. In this thesis we take a closer look at two endogenous peptides that have both been implicated in pain and itch, calcitonin gene-related peptide (CGRP) and endothelin-1 (ET-1).

Calcitonin gene-related peptide CGRP is a 37 amino acid neuropeptide that was first characterized 37 years ago (Amara et al. 1982) and it exists in two different but structurally similar isoforms, CGRPα and CGRPβ, encoded by the genes Calca and Calcb, respectively (Höppener et al. 1985). In the rat, it was found that CGRPα is the main form present in the sensory nervous system (at concentrations 3-6 times that of CGRPβ), including DRG and the dorsal horn of the spinal cord, while CGRPβ is more prominent in the enteric nervous system (Muddhrry et al. 1988). Both forms of CGRP are potent vasodilators (Brain et al. 1985; Van Rossum, Hanisch, and Quiron 1997) and CGRP has been shown to play an important role in the regulation of blood pressure (Gangula et al. 2000; Brain and Grant 2004). It has also been demonstrated that during migraine attacks, large amounts of CGRP are released in the trigeminal system (Bellamy, Cady, and Durham 2006) and by inhibiting the physiological and cellular effects of the peptide, a migraine attack can be prevented (Wrobel Goldberg and Silberstein 2015). Hence, CGRP receptor antagonists and mAbs targeting CGRP have received considerable attention as new migraine treatments. Ac- cording to a new meta-analysis of clinical trials of six CGRP receptor antagonists, the drugs were generally well tolerated and olcegepant was the one that had the best efficacy (Xu and Sun 2019). However, research into olcegepant has been discontinued due to its poor brain bioavailability, and several others have been discontinued as well due to concerns of hepatotoxicity with long- term use. The clinical potential of ubrogepant, rimegepant and atogepant is still under investigation (Agostoni et al. 2019; Xu and Sun 2019). Research in anti-CGRP mAbs has proven more fruitful and currently, three such antibodies are approved for the prophylaxis of migraine: fremanezumab and galcane- zumab, which target the peptide itself, and erenumab, which targets the CGRP receptor (Agostoni et al. 2019). Transgenic and knock-out mice have been generated to further study the pain-mediating function of CGRP in vivo. In a study of knock-out mice, CGRPα-/- animals had lowered responses to capsaicin, formalin and carrageenan injections (Salmon et al. 2001), indicating that CGRPα plays a role in transmitting neurogenic inflammatory pain. The same mouse line had normal responses to acute heat (Salmon et al. 1999). In another study, CGRPα-/- mice

21 showed normal thermal noxious responses in the paw withdrawal and hot- plate tests, but did not develop secondary hypersensitivity when subjected to those same tests after being injected with kaolin/carrageenan in the knee joint to simulate arthritis (Zhang et al. 2001). In a model of neuropathic pain in the rat (spinal nerve ligation), it was observed that CGRP expression was upregulated in DRG (Wang et al. 2010), further supporting the hypothesis that CGRP (as well as SP) contribute to the development and maintenance of such pain (Lee and Kim 2007). In a study by McCoy and colleagues (2013), CGRPα-expressing neurons were selectively ablated from DRG and those mice showed reduced sensitivity to heat and capsaicin. They were also less sensitive to chloroquine- and histamine-induced itch, but not to mechanical stimuli and β-alanine-induced itch, and they were hypersensitive to cold (McCoy et al. 2013). In an earlier study, the same research group demonstrated that around 50% of CGRPα-positive neurons in DRG were TRPV1-positive in mice (McCoy, Taylor-Blake, and Zylka 2012). The main excitatory neurotransmitter in primary afferents, including the TRPV1 population, is glutamate (Lagerström et al. 2010; Rogoz et al. 2012), and it has been established that activation of TRPV1 in sensory nerves results in the release of CGRP (Zygmunt et al. 1999; Assas, Pennock, and Miyan 2014). When glutamatergic transmission was disrupted by removing the vesicular glutamate transporter 2 (VGLUT2) from the TRPV1 population, normal regulation of itch became impaired so mice scratched excessively, and had impaired thermal pain perception as well (Lagerström et al. 2010). In the same mouse line, carrageenan-induced heat hypersensitivity was similar to wild-type mice, but when antagonists to CGRP and SP were administered hyperalgesia did not develop (Rogoz, Andersen, Kullander, et al. 2014). It is therefore suggested that both neuropeptides, SP and CGRP, together with glutamate, are involved in mediating heat hyperalgesia. Taken together, it seems that CGRP plays a role in mediating pain and inflammatory hypersensitivity, and is particularly important in migraine, where successful prophylactic treatments have been based on blocking the peptide. When neurons that express CGRPα in DRG are deleted, acute thermal responses decrease, along with itch mediated through TRPV1-expressing neurons. Thus, it would be informative to see which pain and itch phenotypes are observed in mice where CGRPα is removed from the TRPV1 neuronal population, without obliterating the neurons. This potential pain phenotype is the subject of Paper I of the thesis, where we crossed Calca-loxP mice with a Trpv1-Cre mouse line and evaluated the effects on peripheral pain sensation.

Endothelin-1 Endothelins are 21 amino acid peptides of which three isoforms have been identified, endothelin-1, -2 and -3 (ET-1, ET-2 and ET-3). The first isoform,

22 ET-1, was characterized in 1988 (Yanagisawa et al. 1988) and the other two, ET-2 and ET-3, were described soon after (Inoue et al. 1989). They are only found in vertebrates and are encoded by the genes EDN1, EDN2 and EDN3, respectively (Arinami et al. 1991) and ET-1 is the most widely expressed of the three isoforms (Sokolovsky 1992; Masaki 1993). ET-1 is mostly produced and released by vascular endothelial cells (Yanagisawa et al. 1988) as the name indicates, but its production has been reported in various other cell types, such as keratinocytes (Yohn et al. 1993), vascular smooth muscle cells (Lerman et al. 1991), cardiac myocytes (Suzuki, Kumazaki, and Mitsui 1993), macrophages, mast cells (Ehrenreich et al. 1992; Liu, Yamada, and Ochi 1998) and in DRG and spinal cord neurons (Giaid et al. 1989; Hasue et al. 2005). In contrast to CGRP, the endothelins are potent vasoconstrictors (Inoue et al. 1989); ET-1 has in fact been identified as the most potent endogenous vasoconstrictor in the human body and it plays a physiological role in blood pressure regulation (Davenport et al. 2016). It causes vasoconstriction by interacting with the ETARs on blood vessels (Hay et al. 1993) but paradoxically, it can also cause vasodilation by activating the ETBRs on endothelial cells in an autocrine fashion, which results in the release of vasodilators such as nitric oxide (Verhaar et al. 1998). These actions of ET-1 normally help maintain basal vascular tone and homeostasis, but in hypertension the vasoconstriction mediated by ET-1 via ETAR may contribute to the condition. ETARs are highly expressed in the lung, and currently, three ETR antagonists are approved as treatments against pulmonary arterial hypertension: ambrisentan (ETAR antagonist), bosentan and macitentan (mixed ETAR/ETBR antagonists) (Davenport et al. 2016). Unexpectedly, it was discovered that exogenously administered ET-1 was capable of inducing and potentiating pain and itch in human volunteers (Ferreira et al. 1989; Dahlof et al. 1990; Namer et al. 2008) as well as in ro- dents (Piovezan et al. 1997; Gokin et al. 2001; Trentin et al. 2006; Gomes, Hara, and Rae 2012). In a similar fashion to the effects on blood vessels, ETAR and ETBR have been shown to mediate opposite effects in noci- and pruriception. Peripherally, ETAR activation transmits pain and itch, while the role of ETBR is less clear, but rather connected to analgesic and antipruritic effects (Khodorova et al. 2003; Trentin et al. 2006; Liang, Kawamata, and Ji 2010). In contrast, when ET-1 is administered in the central nervous system it produces analgesia, which can be mediated by both receptor subtypes (Kamei et al. 1993; Yamamoto et al. 1994; D’Amico, Di Filippo, and Rossi 1997; Hasue et al. 2004). Several different knock-out models of ET-1 have been developed in mice; full knock-outs typically develop severe cardiovascular abnormalities (reviewed in Davenport et al. 2016). When ET-1 was selectively ablated from neurons, the mice were more sensitive to acute, inflammatory and neuropathic

23 pain (Hasue et al. 2005), highlighting that neuronal ET-1 has a role in sup- pressing pain. ETAR antagonists have received considerable attention as potential analgesics, particularly in cancer pain and sickle cell disease; however few clinical trials have been published and no medication has been approved yet for this use (Smith et al. 2014; Davenport et al. 2016). ET-1-induced pruritus has not received the same attention as pain, but ET- 1 has been shown to be an extremely potent pruritogen, active in the picomolar range (Imamachi et al. 2009). The peptide induces “burning itch”, i.e. itch with painful qualities, when injected intradermally in humans (Ferreira et al. 1989; Namer et al. 2008). This was also reflected in the mouse cheek model, where algogens injected into the cheek induce wiping behavior by the forepaws, in- dicative of pain; while pruritogens promote scratching by the hind paws, indicating itch sensation (Shimada and LaMotte 2008). When ET-1 was tested in the cheek model, it resulted in a mixture of both behaviors (Gomes et al. 2012). ET-1 can induce mast cell degranulation, by binding to ETARs expressed on the mast cell surface, causing histamine release (Yamamura et al. 1994a; Matsushima et al. 2004a) and this suggests that ET-1-induced itch could be histaminergic in nature. However, it has been shown that ET-1 and histamine differ in their neuronal itch signaling mechanisms (Imamachi et al. 2009; Liang et al. 2010) and H1 antagonists are not effective in attenuating ET-1- induced scratching (Gomes et al. 2012). Furthermore, the extreme potency of ET-1 indicates that it is capable of activating neurons directly (Imamachi et al. 2009). In Paper IV, we investigate how mast cell proteases can affect ET- 1-induced scratching behavior in mice.

The effects of CGRP and ET-1 on immune cells Besides the direct role of CGRP as a neurotransmitter, it has also been established that it is an important mediator in the crosstalk between the nervous and immune systems (Assas et al. 2014). ET-1 is an endothelium-derived peptide with neuropeptide characteristics, and has the ability to affect immune cells as well (Matsushima et al. 2004a; Elisa et al. 2015). CGRP is released from TRPV1-positive peptidergic neurons when they are activated (Zygmunt et al. 1999; Assas et al. 2014), and binds to its receptor components calcitonin receptor-like receptor (CRLR) and the receptor activity-modifying protein 1 (RAMP1), which are found on numerous immune cells, including macrophages (Fernandez et al. 2001), T lymphocytes (Mikami et al. 2011, 2012), dendritic cells (Mikami et al. 2011) and mast cells (Eftekhari et al. 2013). The effects of CGRP on immune cells include regulation of motility and migration of T cells, dendritic cells and mast cells (Levite 2000; De Jonge et al. 2003; Mikami et al. 2011) and regulating production of cytokines such as tumor necrosis factor α (TNFα), and the interleukins IL-4, IL-6, IL-9, IL-10 and IL-17 (Fernandez et al. 2001; Altmayr, Jusek, and

24 Holzmann 2010; Mikami et al. 2011, 2012; Jusek et al. 2012). CGRP can inhibit mast cell degranulation (Feng et al. 2010) but is none the less required (together with SP) for normal mast cell-driven skin inflammation (Siebenhaar et al. 2008). In contrast to CGRP, ET-1 can induce mast cell degranulation, as previously mentioned (Yamamura et al. 1994a, 1994b; Matsushima et al. 2004a). Furthermore, it enhances mast cell production of inflammatory cytokines such as TNFα and IL-6 (Matsushima et al. 2004a). ET-1 also has a similar effect on macrophages, where it stimulates TNFα production (Ruetten and Thiemermann 1997) and on T cells and neutrophils, where it stimulates production of pro-inflammatory mediators such as interferon gamma (IFNγ; Elisa et al. 2015). Macrophages, neutrophils, dendritic cells, B and T lymphocytes, and monocytes have all been shown to express ETRs (Ruetten and Thiemermann 1997; Guruli et al. 2004; Mencarelli et al. 2009; Elisa et al. 2015), which suggests that ET-1 plays a role in immunomodulation, which mainly seems related to pro-inflammatory effects. Neurogenic inflammation is a term used to describe the inflammation that develops as a result of neuropeptide release from activated sensory nerves, and the effects of those neuropeptides on nearby cells, immune cells in particular (Steinhoff et al. 2003, see Figure 2). Since mast cells are frequently in close contact with sensory nerves in the skin, possess receptors for neuropeptides and can release a variety of substances that activate neurons, such as histamine and tryptase, they are of particular interest with regards to cutaneous neurogenic inflammation.

Figure 2. Neurogenic inflammation and examples of mast cell protease involvement. Mast cell degranulation causes release of e.g. histamine and proteases. Tryptase activates PAR2 on sensory nerve endings and this causes release of CGRP and SP, which contribute to vasodilation and edema. Tryptase can degrade CGRP and chymase can degrade SP. SP stimulates mast cell degranulation through the MRGPRX2 and NK1R receptors; CGRP inhibits SP degradation and enhances its release. Mast cell mediators stimulate the release of neuropeptides and sensitize nerves, the transmitters bradykinin and endothelin-1 are degraded by chymase and carboxypeptidase, respectively. PAR2 activation ultimately induces central transmission of pain and itch. Abbreviations: ATP, adenosine triphosphate; PAR2, protease-activated receptor 2; CGRP, calcitonin gene-related peptide; SP, Substance P; MRGPRX2, Mas-related G protein-coupled receptor X2; NK1R, neurokinin-1 receptor. Adapted from Steinhoff et al. (2003).

26 Mast cells Mast cells are granulated immune cells that are found in large numbers throughout the body, especially in skin and mucosal surfaces, where they serve as the immune system’s first line of defense against pathogens and other external invasions. They are derived from bone marrow progenitor cells and have traditionally been divided into two groups based on their distribution in tissue, granule content and function: connective tissue mast cells (CTMCs) and mucosal mast cells (MMCs) (Mekori 2005). They are often found in close proximity to peptidergic sensory nerves and blood vessels (Dimitriadou et al. 1994; Botchkarev et al. 1997) and are key effector cells in allergic reactions. Mature mast cells can contain 500-1000 granules filled with pre-formed mediators such as biogenic amines, proteogly- cans, cytokines and proteases, that they can rapidly release into the extracellular environment when activated (Lagunoff, Martin, and Read 1983; Wernersson and Pejler 2014). This release of granule content is called degranulation and many of the mediators possess inflammatory properties. Mast cell activation can also result in the de novo production of diverse mediators (Wernersson and Pejler 2014). Mast cells can be activated by various stimuli, most commonly by ligands that bind to specific surface-bound receptors on the cell membrane. In more rare cases, such as in mast cell activation syndromes, the cells can also be activated by stress or physical stimuli, such as exercise, temperature changes, sunlight and pressure (Akin 2017; Radonjic-Hoesli et al. 2018). Here we will take a closer look at the receptor-mediated activation of mast cells.

Mast cell receptor activation The classical and best studied form of mast cell activation is the allergic cascade, when IgE binds to the high-affinity receptor FcεRI and crosslinks it, resulting in phosphorylation of signaling proteins that leads to degranulation, secretion and synthesis of various mediators (Jouvin et al. 1994; Turner and Kinet 1999). IgE-mediated activation can be inhibited by immunoglobulin G (IgG) that binds to the FcγRI and FcγRIII receptors (Daëron et al. 1995), but IgG can also induce degranulation by binding to the FcγRIII receptor (Katz and Lobell 1995). Toll-like receptors (TLRs) 2, 3, 4, 6, 7, 8 and 9 are found on mast cells (Takeda et al. 2003; Matsushima et al. 2004b). The activation of TLRs on mast cells by pathogen-derived proteins, DNA/RNA and cell wall components usually results in the production of cytokines, prostaglandins and leukotrienes but not degranulation (Marshall, King, and McCurdy 2003). Peptides such as ET-1, SP and vasoactive intestinal peptide (VIP) can cause mast cell degranulation and chemokine production by binding to GPCRs on mast cells. ET-1 activates ETARs, and until recently it was presumed that SP

27 acted on the neurokinin-1 receptor (NK1R) and VIP on VIP receptor type 2 (VPAC2) (Yamamura et al. 1994b; Paus et al. 1995; Kulka et al. 2008). Inter- estingly, it has been demonstrated that the actions of SP and VIP are also mediated by another receptor, the Mas-related G protein-coupled receptor X2 (MRGPRX2) (Tatemoto et al. 2006).

The MRGPRX2 receptor The MRGPXR2 receptor belongs to the Mas-related family of GPCRs and its expression was first identified in DRG (Robas, Mead, and Fidock 2003). Out- side the DRG, the receptor is highly and selectively expressed in CTMCs but not in MMCs (Tatemoto et al. 2006; Motakis et al. 2014) and its activation has been shown to induce non-IgE-dependent mast cell degranulation (Tatemoto et al. 2006; Meixiong et al. 2019). Various small, basic molecules, termed basic secretagogues, can activate MRGPRX2. The synthetic compound 48/80, a potent mast cell degranulator (Barrett et al. 1985), has been shown to mediate its effects via MRGPRX2 (Tatemoto et al. 2006; Kashem et al. 2011). Other examples of basic secretagogues include various peptides, such as antimicrobial host defense peptides like the cathelicidin LL-37, neuropeptides (as previously mentioned) and various peptidergic as well as small molecule drugs (Tatemoto et al. 2006; Subramanian et al. 2013; McNeil et al. 2015). McNeil and colleagues (2015) discovered that several approved drugs could activate MRGPRX2 and thereby cause pseudo-allergic drug reactions, which can in severe cases lead to ana- phylactic shock. This indicates that MRGPRX2 antagonists could be a potential treatment against these drug-induced reactions. In the same study, it was demonstrated that out of 22 potential candidates in the mouse, it is the Mrgpr member b2 (Mrgprb2) receptor that is the murine ortholog to MRGPRX2. Since MRGPRX2 can be activated by neuropeptides, SP in particular, it has been suggested that the receptor might play a detrimental role in neurogenic inflammation (Subramanian, Gupta, and Ali 2016). In addition, activation of mast cells by antimicrobial peptides results in the release of various itch mediators such as IL-31, IL-6 and leukotriene C4 (Niyonsaba et al. 2010). Since it has been demonstrated that these antimicrobial peptides act on the MRGPRX2 receptor (Subramanian et al. 2013), it becomes an interesting target to consider in non-histaminergic, mast cell-mediated pruritus. This is further supported by new evidence that shows that Mrgprb2 activation results in mast cell degranulation that differs considerably from the one achieved via the classical IgE-FcεRI crosslinking (Meixiong et al. 2019). According to the study, Mrgprb2 activation results in the release of tryptase in favor of the mon- oamines histamine and serotonin, and thus promotes itch in a different fashion than classical IgE-mediated itch (Meixiong et al. 2019). Interestingly, compound 48/80-induced degranulation was shown to result in activation of neurons responsive to β-alanine, chloroquine and serotonin, rather than histamine-

28 responsive neurons (Meixiong et al. 2019). Hence, it might be prudent to re- consider the classification of compound 48/80 as an inducer of histaminergic itch exclusively; the Mrgprb2 activation does result in histamine release to some extent but it might only be partly responsible for the resulting itch.

Immunomodulatory properties of oligonucleotides Oligonucleotides are small RNA or DNA fragments, and several studies have shown that they can have immunosuppressive properties. Oligonucleotides suppress the pro-inflammatory cytokine IL-8 in keratinocytes in vitro and reduce inflammation when topically applied in a contact hypersensitivity mouse model (Dorn et al. 2007). Another study on keratinocytes showed that oligonucleotides could inhibit TLR3-mediated cytotoxicity induced by poly I:C (Grimstad et al. 2012). Single-stranded oligonucleotides (ssONs) also blocked poly I:C-induced responses in human monocyte-derived dendritic cells (moDCs) in vitro, and had anti-inflammatory action in macaques in vivo when administered intranasally (Sköld et al. 2012). Similar action of ssONs was observed when injected in macaque skin (Järver et al. 2018) and the same study showed that ssONs were able to attenuate TLR3, TLR4 and TLR7 signaling in moDCs by inhibiting endocytic pathways, the inhibition was concentration- dependent and a certain oligonucleotide length was required for effect. To summarize, ssONs can have anti-inflammatory action in vivo by different routes of administration and the effects are mediated by interfering with TLR signaling. The effects of ssONs on mast cells and itch have not been investigated however, and that is the topic of Paper III of the thesis, where we look at the effects of ssON on mast cell degranulation and itch, and if these effects are mediated by interfering with the MRGPRX2 receptor.

Mast cell proteases Mast cells contain a large amount of proteases within their granules, it has been estimated that they comprise up to 35% of the total cell protein content (Schwartz, Bradford, et al. 1987; Schwartz, Irani, et al. 1987). Some of the proteases are mast cell-specific, i.e. not expressed in any other cells. The proteases are traditionally divided into three groups based on the site at which they prefer to cleave peptide bonds; chymases, tryptases and carboxypeptidase (Pejler et al. 2007). Chymases and tryptases belong to the family of serine proteases, as they contain a reactive serine side chain (Reynolds et al. 1990). They hydrolyze bonds within the peptide chain of their substrates and are therefore classified as endopeptidases. Chymase has chymotrypsin-like cleavage capabilities and cleaves after large hydrophobic amino acid residues, such as tryptophan, tyrosine and phenylalanine (Powers et al. 1985). Tryptase has trypsin-like cleavage activity, i.e. preferentially cleaves after lysine-arginine residues (Huang et

29 al. 2001). Carboxypeptidase is a Zn-dependent metalloprotease which cleaves amino acid residues (preferentially aromatic) at the C-terminal end of peptide chains and is therefore an exopeptidase (Goldstein et al. 1989; Galli et al. 2015). In mice, these proteases are commonly referred to as mouse mast cell proteases (mMCPs), and in Papers II, IV and V of the thesis, we look into the role of those proteases in itch and tissue pain responses.

Chymase Five mast cell chymases have been identified in mice, mMCP1, mMCP2, mMCP4, mMCP5 and mMCP9, while humans only express one mast cell chymase (Caughey, Zerweck, and Vanderslice 1991; Pejler et al. 2007). mMCP1 and mMCP2 are found exclusively in MMCs while mMCP4 and mMCP5 are only expressed in CTMCs (Reynolds et al. 1990; Huang, Blom, and Hellman 1991; Chu, Johnson, and Musich 1992). mMCP9 is specifically expressed by mast cells of the uterus (Hunt et al. 1997). Despite the initial classification as a chymase, mMCP5 was later found to have elastase-like cleavage capabilities (Kunori et al. 2002). Based on the similar tissue distribution and cleavage specificities, mMCP4 is considered to be the closest functional homolog to human mast cell chymase (Andersson, Karlson, and Hellman 2008). A variety of substrates have been identified for mast cell chymases (Pejler et al. 2007; Pejler 2019), some of which are involved in inflammation, pain and itch; they have been summarized in Table 1. As can be seen in Table 1, mast cell chymase can have anti- inflammatory action by cleaving inflammatory mediators, and can also act in pro-inflammatory fashion by cleaving precursors of inflammatory mediators and thereby generate the active substance. Accordingly, mast cell chymase has been linked to both protective and harmful effects in certain inflammatory diseases and conditions. It has been demonstrated that mast cell chymase can have protective effects in severe asthma in humans (Balzar et al. 2005) and mMCP4 can have the same effects in allergic airway inflammation in mice (Waern et al. 2009, 2013). mMCP4 has been shown to be harmful in induced auto-immune arthritis in mice (Magnusson et al. 2009) and chymase has been postulated to have harmful effects in atopic dermatitis (AD), as high levels of chymase have been observed in the skin of chronic AD patients (Badertscher et al. 2005). Further- more, mMCP4 inhibition has been shown to suppress itch and inflammation in mouse AD models (Imada et al. 2002; Terakawa et al. 2008). Also, human chymase induces an allergic skin reaction in mice when injected (Tomimori et al. 2002), mMCP4 together with mMCP5 contributes to skin inflammation after burn injury in mice (Younan et al. 2010; Bankova et al. 2014) and dog chymase potentiates histamine-induced wheal formation in an allergic dog model (Rubinstein et al. 1990).

30 Tryptase Four mast cell tryptases have been identified in mice, mMCP6, mMCP7, mMCP11 and mouse transmembrane tryptase. mMCP11 and mouse transmembrane tryptase are mainly expressed during the early stages of mast cell development (Wong et al. 2004) while mMCP6, found in CTMCs, is considered the most similar to the human main form, β-tryptase (Hallgren et al. 2000). The commonly used mouse line C57BL/6J does not express mMCP7 (Ghildyal et al. 1994). Similar to chymase, tryptase is capable of activating pathways that induce inflammation and itch but can also cleave pro-inflammatory substrates, as summarized in Table 1. In contrast to chymase, there is substantial evidence that tryptase is harmful in bronchoconstriction and airway inflammation, based on in vivo animal models (Clark et al. 1995; Molinari et al. 1996; Wright et al. 1999; Rice et al. 2000; Oh et al. 2002; Sylvin et al. 2002). Therefore, mast cell tryptase inhibitors have received considerable attention as potential new asthma therapy agents in humans (Cairns 2005), although research efforts have not resulted in any new approved medications to date (Pejler 2019). Just as chymase, tryptase has been postulated to be harmful in arthritis, as high levels of mast cell tryptase have been observed in rheumatoid knee lesions and synovial fluid of osteoarthritis patients (Tetlow and Woolley 1995; Buckley, Gallagher, and Walls 1998), and PAR2 activation by tryptase contributes to joint inflammation in mice (Kelso et al. 2006). Furthermore, tryptase-mediated PAR2 activation induces intestinal inflammation in mice (Cenac et al. 2002) and potent itch is induced via the same mechanism when mice are injected intradermally with tryptase (Ui et al. 2006). Finally, it has been reported that tryptase is up-regulated in the skin of AD, psoriasis and eczema patients (Harvima et al. 1990; Jarvikallio et al. 1997) and it has been suggested that this contributes to the skin inflammation (Namazi 2005). Tryp- tase has not been linked to protective effects in inflammatory conditions in vivo, despite the potential to cleave the pro-inflammatory mediators CGRP and VIP in vitro (Caughey et al. 1988; Tam and Caughey 1990).

Carboxypeptidase Only one carboxypeptidase has been identified to date, carboxypeptidase A3 (CPA3) and in mice, as well as in humans, it is expressed only in CTMCs (Reynolds et al. 1989). Its known cleavage profile is rather narrower than those of chymase and tryptase; identified inflammatory, itch and pain mediators that carboxypeptidase cleaves are summarized in Table 1. CPA3 and mMCP5 depend upon each other for storage within mast cell granules, possibly by complex formation, and as a result, Cpa3-/- mice also lack mMCP5 (Feyerabend et al. 2005) and correspondingly, mMCP5-null mice do not express CPA3 (Huang, Šali, and Stevens 1998).

31 To date, in vivo studies of carboxypeptidase function are limited, but studies in mice have shown that it has an important role in host defense against toxins (sarafotoxins), such as those found in snake and honey bee venom (Metz et al. 2006). Interestingly, similar roles in neutralizing toxins have also been reported for chymase and tryptase; mMCP4 is protective against Gila monster and scorpion venom in vivo in mice (Akahoshi et al. 2011) and tryptase from human mast cells could neutralize snake venom from six different species in vitro (Anderson et al. 2018). This could indicate that the mast cell proteases have an evolutionary role in host defense by toxin degradation at different cleavage sites. Sarafotoxins and endothelins are structurally related, and mast cell proteases can protect against ET-1-induced toxicity in mice (Maurer et al. 2004; Schneider et al. 2007). This protection is likely due to CPA3, since it has been reported to break down ET-1 in vitro (Metsärinne et al. 2002), and mice in which CPA3 had been inactivated were more susceptible to ET-1-induced toxicity (Schneider et al. 2007). In the previously mentioned studies, relatively high doses of ET-1 are injected intraperitoneally (i.p.) to mimic sepsis conditions, as it is a potent vasoconstrictor. This induces fatal toxicity in CPA3- deficient animals while wild-type mice recover. In Paper IV we use low amounts of ET-1 and inject it intradermally, to study ET-1-induced itch in mast cell protease-deficient animals.

32 Table 1. Examples of inflammatory, pain and itch mediators cleaved by mast cell proteases. Adapted from Pejler (2019). Mast cell protease Substrate Substrate role References Inflammation, pain, itch Dog chymase SP (Caughey et al. 1988) (via MC degranulation) Inflammation, pain (brady- Bradykinin, (Reilly, Schechter, Human chymase kinin), bradykinin activa- kallikrein and Travis 1985) tion (kallikrein) Human chymase, Precursor of ET-1 (pain, (Nakano et al. 1997; Big-ET-1 mMCP4 itch) Houde et al. 2013) Human chymase, Analgesia, inflammation, (Goldstein, Leong, human Neurotensin itch and Bunnett 1991) carboxypeptidase Precursor of IL-18 Human chymase Pro-IL-18 (Omoto et al. 2006) (inflammation) Human chymase IL-6, IL-13 Inflammation (Zhao et al. 2005) Precursor of IL-1β Human chymase Pro-IL-1β (Mizutani et al. 1991) (inflammation) Human chymase, (Waern et al. 2013; IL-33 Itch, inflammation mMCP4 Fu et al. 2017) (Piliponsky et al. mMCP4 TNF Inflammation 2012) mMCP4 HMGB1 Inflammation (Roy et al. 2014) Kininogen, Inflammation, pain (via kal- Human tryptase (Imamura et al. 1996) prekallikrein likrein/bradykinin pathway) Dog tryptase, (Caughey et al. 1988; Inflammation, itch (via MC Tam and Caughey human tryptase, VIP degranulation) 1990; Akahoshi et al. mMCP4 2011) Inflammation, pain, (Tam and Caughey Human tryptase CGRP 1990; Walls et al. itch modulation 1992) (Cenac et al. 2002; Ui Human tryptase PAR2 Itch, inflammation et al. 2006) Precursor of NGF (modula- Human tryptase Pro-NGF tory effects in inflamma- (Spinnler et al. 2011) tion, heat sensitization) Rat (Metsärinne et al. carboxypeptidase, ET-1 Pain, itch 2002; Schneider et al. mouse CPA3 2007) Abbreviations: SP, Substance P; MC, mast cell; ET-1, endothelin-1; IL, interleukin; mMCP4, mouse mast cell protease 4; TNF, tumor necrosis factor; HMGB1, high mobility group protein B1; VIP, vasoactive intestinal peptide; CGRP, calcitonin gene-related peptide; PAR2, protease-activated receptor 2; NGF, nerve growth factor; CPA3, carboxypeptidase A3.

33 Tools to study in vivo function of mast cell proteases in mice Besides directly injecting mast cell proteases, their effects in vivo can be studied by using protease inhibitors, and transgenic and knock-out mice. The use of protease inhibitors in animal models is limited, since all such inhibitors have been developed to specifically inhibit the human proteases and may thus not be specific enough for the animal homologs or have unexpected off-target effects (Galli et al. 2015). Therefore the use of genetically modified mice has been preferred, and to date mice deficient in the following mast cell proteases have been reported: mMCP1 (Wastling et al. 1998), mMCP4 (Mcpt4-/-; Tchougounova, Pejler, and Åbrink 2003), mMCP5 (Younan et al. 2010), mMCP6 (Mcpt6-/-; Shin et al. 2008) and CPA3 (Cpa3-/-; Feyerabend et al. 2005). A mouse expressing enzymatically inactive CPA3 has also been generated, Cpa3Y356L,E378A (Schneider et al. 2007) and it has been found that mice of the C57BL/6J background in- herently lack mMCP7 (Ghildyal et al. 1994). Furthermore, a triple knock-out lacking mMCP4, mMCP6 and CPA3 has been reported (Mcpt4-/-/Mcpt6-/- /Cpa3-/-; Grujic et al. 2013). In Papers II, IV and V, Mcpt4-/-, Mcpt6-/-, Cpa3-/- and Cpa3Y356L,E378A mice, as well as the triple protease knock-out Mcpt4-/-/Mcpt6-/-/Cpa3-/- have been used to evaluate the individual and combined roles of the respective mast cell proteases in itch and tissue-injury pain responses. In addition, the mast cell deficient mouse line Mcpt5Cre+;R-DTA was used in Paper II.

Monoclonal antibodies against pain and itch So far, we have mainly discussed exogenous agents that contribute to pain and itch sensation. There are however a number of available pharmaceutical therapies that act in the periphery to alleviate pain and pruritus. Many of them are well established, such as local anesthetics like lidocaine, which acts by blocking NaV channels (Shah, Votta-Velis, and Borgeat 2018), and topical cortico- steroids, which have widespread anti-inflammatory and immunosuppressive effects and have become the first-line treatment for conditions such as AD (Mayba and Gooderham 2017). New discoveries and better understanding of the relevant pain and itch pathways have opened up the possibilities of better targeted and more efficient therapies with fewer undesirable side effects. In this context we will take a closer look at a few monoclonal antibodies (mAbs) that are directed against specific immunological targets. Generally, their production is based on cloning a unique parent immune cell, generating multiple identical cells that pro- duce an antibody that recognizes a specific epitope of the desired antigen (de St. Groth and Scheidegger 1980).

34 Two types of mAbs have previously been mentioned in the thesis, the mAb against IgE, omalizumab, that can be used in chronic urticaria (Kolkhir et al. 2019) and the mAbs against CGRP and its receptor that are used in migraine prophylaxis (Agostoni et al. 2019). A few mAbs that target the inflammatory cytokine TNFα have been developed: infliximab (Elliott et al. 1994), adalimumab (Kempeni 1999), certoli- zumab pegol (Choy 2002) and golimumab (Zhou et al. 2007). They have been approved for use in various painful and inflammatory conditions, most notably in rheumatoid arthritis and inflammatory bowel diseases (Mitoma et al. 2018) but also in skin conditions such as psoriasis (Campanati et al. 2018), where the anti-inflammatory effects also help to decrease pruritus. Dupilumab is a mAb that targets the α subunit of the IL-4 receptor, and its binding inhibits both IL-4 and IL-13 signaling (Wenzel et al. 2013). In 2017, dupilumab was approved for the treatment of moderate-to-severe AD in adults, where it has been shown to reduce disease activity and significantly improve pruritus in a clinically relevant manner (Vakharia and Silverberg 2017). A new study of 20 recalcitrant pruritus patients with different conditions, such as prurigo nodularis, uremic pruritus and lichen planus, reported that all patients had a significant improvement in their itch intensity with dupilumab treatment (Zhai et al. 2019). Dupilumab may very well become the gold standard in moderate-to-severe AD treatment (Vakharia and Silverberg 2017) and it appears to have a promising potential in other types of pruritus as well, although further studies are needed. Several other mAbs are currently under investigation for the treatment of AD, since it is a condition with a hetero- genous phenotype where multiple inflammatory mediators can be involved. Most of the mAbs target interleukins, two promising examples against pruritus in AD are tralokinumab, an IL-13 mAb, and the IL-31 receptor mAb, nemol- izumab (Vakharia and Silverberg 2017). Secukinumab is an antibody against IL-17A (Hueber et al. 2010) that is approved for use in plaque psoriasis, psoriatic arthritis and ankylosing spondylitis (Blair 2019); in psoriasis of the scalp, for instance, it has been reported to improve pain, itch, scaling and quality of life (Feldman et al. 2017). Crizanlizumab is a novel antibody against P-selectin, a cell adhesion molecule that initiates the adhesion of leukocytes to the endothelium in vaso-oc- clusive crisis, a painful complication of the disease sickle cell anemia (Ataga et al. 2017). Crizanlizumab has been deemed to be effective in preventing such crises (Ataga et al. 2017; Kutlar et al. 2019), and is expected to enter regulatory review in 2019 (Kaplon and Reichert 2019). Currently, over 80 mAbs have been approved for therapeutical purposes and many of them target cancers and inflammatory conditions (Kaplon and Reichert 2019). 62 new mAbs are reported to be in the final stages of clinical studies (Kaplon and Reichert 2019). Due to their specificity mAbs are usually well tolerated, the risk of drug-drug interactions is low, and they generally have long half-lives which permits less frequent administration (Vakharia and

35 Silverberg 2017). However, they are expensive compared with older medications, so their use is often limited to those patients that do not respond ade- quately to other therapies. The emergence of more affordable biosimilars, the generic equivalents to biopharmaceutical agents like mAbs, might change that.

36 Aims

The overall aim of this work is to elucidate several factors that contribute to the peripheral regulation of acute pain and itch, with a particular focus on the role of CGRP and mast cells. The specific aims of each paper were:

Paper I To investigate the role of CGRPα within the TRPV1-expressing neuronal population in peripheral pain of both somatic and visceral origin.

Paper II To evaluate the role of mouse mast cells and mast cell proteases mMCP4, mMCP6 and CPA3 in inflammatory pain and tissue injury behavioral responses.

Paper III To analyze the effects of a single-stranded nucleic acid on itch and mast cell degranulation.

Paper IV To evaluate the individual and combined effects of mouse mast cell proteases mMCP4, mMCP6 and CPA3 in endothelin-1-induced itch.

Paper V To evaluate the individual and combined effects of mouse mast cell proteases mMCP4, mMCP6 and CPA3 in itch of both histaminergic and non-histaminergic origin.

37 Results and discussion

Paper I It has been well established that the TRPV1 ion channel and TRPV1-expressing primary afferents play an important role in thermal nociception, inflammation-induced hypersensitivity and itch transmission (Caterina et al. 2000; Davis et al. 2000; Imamachi et al. 2009; Lagerström et al. 2010; Mishra et al. 2011). Furthermore, it has been shown that CGRPα-expressing neurons play a role in thermal sensation, pain and itch (McCoy et al. 2012, 2013) and that CGRP, together with glutamate, mediates heat hypersensitivity within the TRPV1 population (Rogoz, Andersen, Kullander, et al. 2014). It has been reported that 50-70% of CGRP-expressing neurons are TRPV1-positive (McCoy et al. 2012; Rogoz, Andersen, Kullander, et al. 2014) and therefore it is of considerable interest to look into the role of CGRP within the TRPV1 population with regards to pain transmission. This is particularly interesting with regards to visceral pain, as information about the role of CGRP in such transmission is limited. In this paper, CGRPα was selectively removed from the TRPV1 population, by cross-breeding CGRPα-mCherrylx/lx mice with Trpv1-Cre-expressing mice. mCherry is a red fluorescent protein reporter that was incorporated to facilitate the visualization of CGRPα expression. Behavioral tests were then performed to investigate the role of CGRPα within the TRPV1 population in peripheral pain transmission, of both visceral and somatic origin, as well as olfaction. Furthermore, immunohistochemistry and quantitative polymerase chain reaction (qPCR) were employed to evaluate CGRP expression, and electrophysiology was used to investigate its properties in mechanosensitivity of the colon. A major finding of the study was that CGRPα-mCherrylx/lx;Trpv1-Cre mice were less sensitive to the pain accompanied with i.p. injection of acetic acid, as demonstrated by the fact that they spent less time immobile after the injection than their controls, Cre-negative littermates. It is well known that animals experiencing high levels of pain tend to stay immobile (Gebhart et al. 2009; Cho et al. 2013), and in this study, many of the control mice stayed stationary in one corner of the recording cage after the injection. The CGRPα-mCher- rylx/lx;Trpv1-Cre mice also travelled a longer distance in the recording cage after injection, but showed no difference from controls in pain responses commonly quantified in this pain model, i.e. abdominal licking and stretching, and

38 facial wiping. This suggests that measuring levels of immobility and distance travelled could be valid indicators of pain following i.p. acetic acid injection, instead of only quantifying the other pain behaviors previously listed. When looking into mechanosensitivity of the colon using Von Frey hairs and cir- cumferential stretch coupled with electrophysiology, no differences were observed between genotypes. CGRPα-mCherrylx/lx;Trpv1-Cre mice were not different from controls with regards to noxious cold or heat sensation. They did however develop a more pronounced heat hypersensitivity (i.e. it developed earlier and lasted longer) after nerve growth factor β (NGFβ) injection compared with their baseline heat sensitivity values, but there was no significant difference observed between genotypes at any time point. This is in accordance with our previous experiments, showing that CGRP alone is not responsible for noxious cold or heat hypersensitivity, and that instead, SP together with glutamate is responsible for cold sensitivity, and glutamate is required together with CGRP and/or SP for heat hypersensitivity (Rogoz, Andersen, Kullander, et al. 2014; Rogoz, Andersen, Lagerström, et al. 2014). Our results in paper I seem to contradict what has been previously reported on the role of CGRPα in thermal transmission (McCoy et al. 2013), however, an important difference is that in the McCoy experiment the CGRPα-expressing neurons were ablated and thus the dominant excitatory transmitter glutamate, responsible for noxious heat transmission (Lagerström et al. 2010) is also removed. A qPCR analysis was performed to confirm that the Cre-LoxP recombina- tion had been successful in reducing CGRPα mRNA expression in the DRG. Indeed, CGRPα-mCherrylx/lx;Trpv1-Cre mice had only around 25% of the expression seen in controls. Surprisingly, when analyzing DRG and colon from CGRPα-mCherrylx/lx;Trpv1-Cre mice using immunohistochemistry, it was observed that total CGRP immunoreactivity (comprising both CGRPα and CGRPβ) was unaltered compared with controls. This suggests that upon con- ditional removal of CGRPα from the TRPV1 population, up-regulation of CGRP takes place, of either CGRPα from non-TRPV1 neuronal populations or of CGRPβ within the TRPV1 population. Such upregulation of CGRPβ upon deletion of the Calca gene has been reported previously (Huebner et al. 2008). However, our qPCR analysis showed no differences in the relative expression of CGRPβ mRNA in the DRG of CGRPα-mCherrylx/lx;Trpv1-Cre mice compared with controls. This discrepancy could be explained by the fact that the TRPV1 population holds a greater proportion of the total CGRPα population than it holds of the total CGRPβ population. This means that CGRPβ could be upregulated but it would be harder to detect a significant difference with qPCR due to its lower expression levels compared with the ubiquitous CGRPα. Taken together, the data from paper I indicate that CGRPα-mediated transmission within the TRPV1 population is important in visceral pain perception, particularly in voluntary movements but not in acute reflex responses such as

39 writhing and abdominal licks. Based on our findings, the individual role of CGRPα within the TRPV1 population is not vital in somatic thermal nociception or inflammatory heat hypersensitivity, but plausible upregulation of CGRP upon the removal of the Calca gene may complicate the interpretation of those results.

Paper II Mast cells have generally been considered important components in inflammatory and protective responses against exogenous threats (Galli, Grimbaldeston, and Tsai 2008; Voehringer 2013). Since they contain a large amount of proteases that have been associated with both protective and inflammatory properties, we considered it of interest to investigate the involvement of three proteases (mMCP4, mMCP6 and CPA3), as well as mast cells in general, in the acute inflammation and tissue-injury pain responses resulting from formalin injection. Further, we looked at the role that mast cells play in noxious heat sensation and inflammatory heat hypersensitivity induced by NGFβ injection. To achieve this, we performed the formalin test on the individual mouse knock-outs of the aforementioned proteases, and mast cell deficient mice (Mcpt5Cre+;R-DTA) underwent the same test, as well as noxious heat and heat hypersensitivity tests. In the published paper, the protease knock-out mice are referred to as mMCP4-/-, mMCP6-/- and CPA3-/-, but here they are referred to as Mcpt4-/-, Mcpt6-/- and Cpa3-/-, respectively, in order to harmonize with the presentation in papers IV and V. Mcpt4-/- mice did not differ significantly from wild-type controls at any time interval during the 60 minutes of the formalin test, but did show a slight trend for exhibiting higher pain responses during the later stages of the inflammatory phase (45-60 minutes). The pain induced by formalin in both the initial and secondary phase is mediated by activation of TRPA1 (McNamara et al. 2007), and it has been demonstrated that bradykinin, released as a result of tissue injury, mediates formalin-induced pain (da S Emim et al. 2000) and activates TRPA1 (Bandell et al. 2004; Meotti et al. 2016). mMCP4 degrades bradykinin in vitro (Reilly et al. 1985) so it is an interesting possibility that mMCP4 exerts its slight protective function in the formalin test by cleaving bradykinin, thus attenuating TRPA1 activation. Furthermore, it has been shown that IL-33 is released from necrotic cells upon tissue injury (Cayrol and Girard 2009; Lüthi et al. 2009), has a role in mediating formalin-induced pain (P. Han et al. 2013) and is degraded by mMCP4 (Waern et al. 2013; Roy et al. 2014). Possible protective effects of mMCP4, although not significant, could thus also be due to IL-33 cleavage. Contrary to Mcpt4-/- mice, Mcpt6-/- mice demonstrated slightly lower pain responses than controls during the inflammatory phase of the formalin test,

40 although the trend was not statistically significant. This small effect could also be attributed to bradykinin-induced activation, since it has been shown that human tryptase can cleave the bradykinin precursor, kininogen, to generate active bradykinin (Imamura et al. 1996). Furthermore, tryptase can activate the precursor prekallikrein to generate kallikrein, the serine protease responsible for converting kininogen into bradykinin (Imamura et al. 1996). Less contribution to bradykinin production could therefore be an explanation of the trend for slightly lower pain responses observed in Mcpt6-/- mice. Cpa3-/- mice did not differ from controls in their pain behavior. This is per- haps not surprising since carboxypeptidase has to date not been shown to be involved in inflammation (Caughey 2016), but mainly has protective effects through venom degradation (Metz et al. 2006). CPA3 does cleave the algogen ET-1, but blocking ETAR and ETBR did not have an effect on formalin-induced pain (Piovezan et al. 1997). CPA3-deficient mice also lack the elastase mMCP5 (Feyerabend et al. 2005), which contributes to skin inflammation after burn injury in mice, together with mMCP4 (Younan et al. 2010). However, the mMPC5-deficiency does not appear to have an effect in the formalin-induced pain responses observed in the Cpa3-/- mice. The mast cell-deficient mice, Mcpt5Cre+;R-DTA, did not differ significantly from their Cre-negative littermate controls at any time point or phase during the formalin test. No differences were observed between genotypes either in noxious heat sensation or in the NGFβ-induced heat hypersensitivity test. In- terestingly, the controls failed to develop hypersensitivity compared with their baseline heat sensitivity values, although a trend could be observed, but the Mcpt5Cre+;R-DTA mice did develop significant hypersensitivity at one time point, four hours after NGFβ injection. This was somewhat surprising since mast cells have been considered important participants in initiating inflammatory responses. However, this result is in accordance with a previous study showing that mast cells were not required for the development of heat or mechanical hypersensitivity induced by NGFβ or complete Freund’s adjuvant (Lopes et al. 2017). It has also recently been suggested that mast cells mostly play a role in IgE-mediated inflammation but may have a negligible role in other types of inflammation (Maurer et al. 2019). Taken together, the data indicate that mast cells and mast cell proteases do not play a significant role in the pain responses observed in the tissue injury and acute inflammation resulting from formalin injection. Although not significant, mMCP4 could potentially have protective effects in the pain responses observed in the secondary (inflammatory) phase of the formalin test, while mMCP6 might have pro-inflammatory properties. Based on our data, mast cells do not play a significant role in noxious heat sensation or in inflammatory heat hypersensitivity induced by NGFβ.

41 Paper III The Mas-related G protein-coupled receptor X2 (MRGPRX2) is highly and selectively expressed on human skin mast cells (Motakis et al. 2014) and has been reported to modulate itch through non-IgE-dependent mast cell degranulation. MRGPRX2 and the mouse ortholog Mrgprb2 can be activated by various basic cationic substances, such as neuropeptides, antimicrobial peptides, certain drugs and the mast cell activating compound 48/80 (McNeil et al. 2015; Subramanian et al. 2016). Oligonucleotides have been reported to re- press certain immunological responses (Dorn et al. 2007; Grimstad et al. 2012). It was previously demonstrated that a novel 35 base-long single- stranded oligonucleotide (ssON) was able to modulate cytokine production in skin cells in vitro and in macaques in vivo (Sköld et al. 2012; Järver et al. 2018). In this study we looked at the effects of ssON on MRGPRX2/Mrgprb2 signaling and the implications for mast cell degranulation and itch of both histaminergic and non-histaminergic origin. To investigate the effects of ssON, two different doses were tested (2.5 and 25 µg) together with compound 48/80 (mast cell degranulator with histaminergic properties) or the PAR2/MrgprC11 agonist SLIGRL (non-histaminergic itch) in BALB/c mice. The higher dose of ssON significantly reduced compound 48/80-induced scratching in two separate experiments, while no effects on SLIGRL-induced scratching were observed. By examining skin biopsies of mice injected with compound 48/80, either alone or together with ssON, we saw that ssON inhibited mast cell degranulation induced by compound 48/80, and the level of degranulation seen in ssON-treated animals was com- parable to saline-injected mice. Studies of human mast cells (LAD2 and cord blood-derived mast cells, CBMCs) in vitro revealed that ssON was also able to inhibit degranulation induced by compound 48/80 and the antimicrobial peptide LL-37, but not degranulation caused by the neuropeptide SP or the calcium ionophore A23187. ssON did not have an effect on IgE-mediated degranulation. We determined that the inhibitory effect was specific to ssON by testing another single-stranded oligonucleotide (ON15, 15 base-long) that has previously been shown to be without immunosuppressive properties (Järver et al. 2018). ON15 failed to inhibit compound 48/80-mediated degranulation in both LAD2 cells and CBMCs, thus showing that the inhibition by ssON is not due to common properties of nucleotides. To investigate if ssON had an effect on MRGPRX2 activation, we used a MRGPRX2-transfected human embryonic kidney cell line (HEK293) and used flow cytometry to measure Ca2+ influx as an indicator of cell activation. Compound 48/80, LL-37 and, in contrast with the previous results, SP, all induced substantial Ca2+ influx, which was abolished in all instances when ssON was added. However, ssON did not have an effect on Ca2+ signaling induced by morphine or the MRGPRX2 agonist ZINC-3573 in the transfected cells.

42 Non-MRGPRX2-expressing HEK293 cells were not activated by compound 48/80 or SP, but did have a moderate Ca2+ influx in response to LL-37, which was also inhibited by ssON. It was interesting to note that ssON inhibited SP- induced activation in HEK293-transfected cells but not in LAD2 cells. A possible explanation could be that LAD2 cells also express the NK1R receptor while HEK293 cells do not, and SP has been reported to induce degranulation by activating NK1R (Kulka et al. 2008). Taken together, the above data show that ssON can inhibit compound 48/80-induced scratching in mice by blocking mast cell degranulation. ssON also inhibits human mast cell degranulation instigated by compound 48/80 and LL-37, and this effect is likely mediated by blocking MRGPRX2. However, ssON does not appear to inhibit the activation of MRGPRX2 by all secretagogues, so its effects might be related to specific binding sites on the receptor or particular downstream signaling events.

Paper IV ET-1 is an endogenous peptide that has a potent vasoconstricting function. It also has algogenic and pruritogenic properties that are mediated through ETA receptors (ETARs) found on primary afferents (Gokin et al. 2001; Gomes et al. 2012), and ET-1 has the ability to activate and degranulate mast cells via the same receptor (Yamamura et al. 1994a; Matsushima et al. 2004a). One of the mast cell proteases discussed in Paper II, CPA3, can cleave ET- 1 (Metsärinne et al. 2002) and CPA3-deficient mice have been reported to die of ET-1-induced toxicity while control mice recover (Schneider et al. 2007). Considering this, we thought it of interest to investigate the role of CPA3 in ET-1-induced itch. Furthermore, mast cell chymase and tryptase have been implicated in itch and pruritic skin conditions; chymase is upregulated in AD (Imada et al. 2002; Badertscher et al. 2005), and tryptase can cleave and activate the PAR2 receptor, which is known to mediate itch and inflammation (Ui et al. 2006). In this study, we used mice deficient in mMCP4, mMCP6 and CPA3, as well as mice lacking all three proteases, in order to study their individual and combined effects in ET-1-induced itch. Our first set of experiments revealed that Mcpt4-/- mice did not differ from controls in their scratching behavior but both Mcpt6-/- and Cpa3Y356L,E378A mice scratched significantly more than controls, both with regards to scratching frequency and duration. The Cpa3Y356L,E378A mouse line was initially used since it expresses enzymatically inactive CPA3, however they express fully functional mMCP5 (elastase), while the full CPA3 knock-out expresses neither CPA3 nor mMCP5 since the proteases depend upon each other for granule storage. The Cpa3Y356L,E378A mice thus gave us the opportunity to look at the isolated effects of lacking only CPA3 function.

43 Next, we were interested in seeing how multiple mast cell protease deficiency would affect the ET-1 scratching response. The Mcpt4-/-Mcpt6-/-Cpa3- /- animals exhibited highly elevated levels of scratching after ET-1 injection. We concluded that this was a result of lacking both mMCP6 as well as CPA3, but it was still possible that loss of mMCP5 played some part in this extreme phenotype. The next step was to test the full CPA3 knock-out, Cpa3-/-, and see if their scratching behavior would resemble that of Cpa3Y356L,E378A mice or the more exaggerated behavior of Mcpt4-/-Mcpt6-/-Cpa3-/- animals. The scratching behavior of the Cpa3-/- mice was elevated compared with controls but fol- lowed a similar pattern and magnitude as seen in the Cpa3Y356L,E378A mice, so we concluded that mMCP5 was not a factor in ET-1-induced itch. CPA3 can cleave ET-1 and has been demonstrated to have an important protective function in ET-1-induced toxicity in vivo (Schneider et al. 2007). Therefore we drew the conclusion that the scratching phenotype observed in both CPA3-deficient mouse lines could be explained by higher levels of ET- 1 activation on primary afferents, and possibly on other cells that express ETARs. However, it remained to elucidate the role of mMCP6 in ET-1-induced scratching behavior. Based on our in vitro studies, human lung β-tryptase did not cleave ET-1 directly, so mMCP6 appeared to be involved in another part of the cascade. We saw that the ETAR antagonist BQ-123 was effective in almost completely abolishing ET-1-induced scratching in the first 40 minutes after injection in wild-type mice but the effect was less significant in Mcpt6-/- mice, which indicates that some additional factors contribute to their scratching phenotype. When ET-1 binds to and activates its receptor on primary afferents it causes the release of neurotransmitters such as CGRP and SP into the extracellular space, and around 70% of ETAR-positive primary afferents express CGRP as well (Usoskin et al. 2015). Mast cell tryptase is very efficient in degrading CGRP, thus inactivating its vasodilating action (Tam and Caughey 1990; Walls et al. 1992) and it has also been seen that CGRP plays a role in PAR2- mediated itch (Costa et al. 2008), as well as itch instigated by glutamate deficiency in TRPV1 neurons (Rogoz, Andersen, Lagerström, et al. 2014). It therefore seemed possible that the higher scratching levels seen in Mcpt6-/- mice were to some extent linked to higher extracellular levels of CGRP. We therefore tested injecting Mcpt6-/- mice with either a CGRP antagonist (BIBN4096) or vehicle, prior to ET-1 administration. No differences were observed between treatment groups, except that the antagonist-treated mice dis- played more frequent scratching in the first ten minutes after ET-1 injection. Based on this result, it appears that CGRP does not play a role in propagating ET-1-induced itch, but might rather have a small attenuating role in the early phase after injection. In summary, this study showed us that both mast cell proteases CPA3 and mMCP6 have protective effects in ET-1-induced itch and when both proteases

44 are lacking, the scratching behavior becomes more intense. CPA3 presumably exerts its effects by directly cleaving ET-1 in vivo, while the role of mMCP6 is less clear and probably related to other events in the ET-1-induced cascade, such as neurogenic inflammation or mast cell degranulation. This is further supported by the fact that blocking ETAR was not as efficient in decreasing the scratching behavior in Mcpt6-/- mice as in wild-type controls.

Paper V

In this paper, we continued investigating the role of the mouse mast cell proteases in itch, using the same single and multiple protease knock-out mouse lines as in Paper IV, but instead of ET-1 we looked at other pruritogens of both histaminergic (histamine, compound 48/80) and non-histaminergic (chloroquine, SLIGRL) origin. We saw that the triple-deficient Mcpt4-/-/Mcpt6-/-/Cpa3-/- mice scratched more frequently and spent more total time scratching than wild-type controls in response to histaminergic itch, induced by histamine and compound 48/80. No differences were observed in the frequency or duration of scratching resulting from MrgprA3- and PAR2/MrgprC11-mediated itch, induced by chloroquine and SLIGRL, respectively. When skin samples were analyzed, it was discovered that the Mcpt4-/- /Mcpt6-/-/Cpa3-/- mice had fewer mast cells present in skin than controls, but levels of degranulation were similar between genotypes and a difference was not seen between saline- or histamine-injected mice, indicating that histamine on its own does not cause mast cell degranulation. The reason for fewer mast cells in the skin of Mcpt4-/-/Mcpt6-/-/Cpa3-/- mice is not known, but multiple mast cell protease deficiency affects electrical properties of the mast cell granules and could affect their motility properties as well. The Mcpt4-/-/Mcpt6-/- /Cpa3-/- mice also lack mMCP5, a mast cell protease with elastase-like cleavage properties (Kunori et al. 2002). The multiple protease-deficiency makes it problematic to interpret the results with regards to individual effects of each protease, so the next step was to look into scratching behavior of the single mast cell protease knock-outs, with special emphasis on histaminergic itch. The Mcpt4-/- mice were tested with histamine, compound 48/80 and chloroquine and did not differ from controls in any parameter of their scratching behavior. This was somewhat surprising, as mast cell chymase has been reported to cleave various mediators of inflammation and itch, including SP (Caughey et al. 1988), a neuropeptide that can cause histamine release through mast cell degranulation (Lowman, Benyon, and Church 1988). This indicates that SP is not a factor in the increased histaminergic itch observed in the Mcpt4-/-/Mcpt6-/-/Cpa3-/- mice, or that mMCP4 is not an essential protease in degrading SP in the extracellular environment.

45 When injected with histamine, neither Mcpt6-/- mice nor Cpa3-/- mice differed from controls with respect to their scratching frequency or duration. The Cpa3-/- mice did however have slightly shorter scratching episodes in response to histamine than controls, which can be an indicator of more intense scratching. When injected with compound 48/80, both Mcpt6-/- and Cpa3-/- mice demonstrated more than twice as many scratching episodes in 60 minutes than controls, their total scratching duration was longer and the mean length of scratching episodes was shorter. When the scratching frequency was analyzed over time, it was seen that the knock-outs scratched more often than controls overall. Mcpt6-/- mice scratched more than controls during the middle of the test (between 20 and 40 minutes after injection), and Cpa3-/- mice scratched more during the first 20 minutes of the test as well as in the last 10 minutes. Compound 48/80 is a potent mast cell degranulator (Barrett et al. 1985) and causes the release of numerous mast cell mediators that can activate sensory neurons to transmit itch and release neurotransmitters such as CGRP and SP. As previously mentioned (Paper IV) is mast cell tryptase very efficient in degrading CGRP (Tam and Caughey 1990; Walls et al. 1992). It is therefore possible that the higher scratching levels seen in Mcpt6-/- mice are due to higher extracellular levels of CGRP. However, the CGRP receptor antagonist BIBN4096 was not effective in reducing scratching induced by ET-1 in another study we performed (Paper IV). The precise role of mMCP6 in acute itch remains elusive, it might be involved in either the breakdown of mast cell mediators released in degranulation or in cleaving transmitters consequently released during neurogenic inflammation. A recent study showed that compound 48/80-induced degranulation via the Mrgprb2 receptor results in the preferential release of tryptase (mMCP6) at the expense of histamine and serotonin (Meixiong et al. 2019). Based on this, it would be interesting to see how mMCP6-deficiency affects the degranulation profile of compound 48/80, and if this could provide further explanation of the scratching phenotype observed. The Cpa3-/- mice showed, similar to the Mcpt6-/- mice, enhanced scratching in response to compound 48/80 but not to histamine. CPA3 is mainly involved in degrading sarafotoxins, such as those found in snake and bee venom (Metz et al. 2006) but it can also break down ET-1 (Metsärinne et al. 2002; Schneider et al. 2007) as previously mentioned (Paper IV), which we deemed interesting to consider as a possible mediator in the compound 48/80 itch cascade. ET-1 can be produced by mast cells under certain conditions but its release is considered to be independent of degranulation (Ehrenreich et al. 1992). Cathepsin E, however, is a mast cell protease released upon degranulation (Henningsson et al. 2005) and converts the precursor big-ET-1 into ET-1 in skin, resulting in pruritus (Lees et al. 1990; Andoh et al. 2012). Higher extracellular levels of ET-1 could thus be a possible contributor to the enhanced scratching response -/- in Cpa3 mice, as it not only mediates itch through ETAR (McQueen et al. 2007) but can also degranulate mast cells (Yamamura et al. 1994a). We had

46 also seen that mice deficient in CPA3 were particularly sensitive to ET-1 (Pa- per IV). Based on this, we injected the ETAR antagonist BQ-123 together with compound 48/80 in Cpa3-/- mice and compared with Cpa3-/- mice that only received the pruritogen. No differences in scratching behavior were observed between the treatment groups so we concluded that ETAR activation is not a factor in the scratching induced by compound 48/80 in Cpa3-/- mice. This is in accordance with a previous study showing that blocking ET receptors does not affect compound 48/80-induced scratching in Swiss mice (Trentin et al. 2006). Taken together, the results of paper V indicate that mMCP6 and CPA3 have a protective role in scratching behavior resulting from compound 48/80 prov- ocation, and these protective effects are probably related to other mast cell compounds than histamine, since histamine alone did not evoke scratching that differed from controls. The extensive mast cell degranulation caused by compound 48/80 may trigger multifaceted activation of sensory neurons, resulting in neurotransmitter release and neurogenic inflammation, in which mMCP6 and CPA3 may have protective roles. The enhanced response of Mcpt4-/-/Mcpt6-/-/Cpa3-/- mice to compound 48/80 is likely due to the dual absence of mMCP6 and CPA3, but the higher scratching levels seen with histamine alone were not observed in any of the single knock-outs, indicating that they can be due to the combined, unknown effects of lacking multiple mast cell proteases.

47 Summary and future perspectives

In this thesis I have focused on several factors that can influence pain and itch sensation in the periphery, with special emphasis on connective tissue mast cells and their proteases, as well as CGRP within the TRPV1 neuronal population. We have seen that while CGRPα has a role in transmitting visceral pain within the TRPV1 population, its targeted deletion from the population does not affect somatosensory pain with regards to neither cold, heat nor inflammatory heat hypersensitivity. The immunohistochemical data from the study suggested that upregulation of either CGRP isoform, α or β, had taken place within the DRG of αCGRP-mCherrylx/lx;Trpv1-Cre mice. Our qPCR data, however, did not support this observation, possibly due to the relatively large differences in expression levels between the different CGRP isoforms within the DRG, where CGRPα is the more prevalent form. If upregulation does indeed takes place, the αCGRP-mCherrylx/lx;Trpv1-Cre mice are not an ideal model to study the effects of CGRPα in the periphery. Better information might be gathered using agonists and antagonists of the CGRP receptor, together with behavioral testing, or by employing in vitro methods. Somewhat surprisingly, we saw that mast cells and mast cell proteases were not essential in tissue injury-induced pain responses as evaluated in the formalin test. Furthermore, the absence of mast cells did not prevent the development of inflammatory heat hyperalgesia induced by NGFβ. This supports the notion that mast cells play a smaller role than previously believed in IgE- independent inflammatory responses, as recently suggested (Maurer et al. 2019). Since the characterization of the mast cell-specific MRGPRX2 and its mouse ortholog Mrgprb2 as important receptors for pseudo-allergic reactions (McNeil et al. 2015) that can be activated by a variety of different molecules, they have received considerable attention as interesting targets against mast cell-mediated responses. Using a novel single-stranded oligonucleotide (ssON), we could see that compound 48/80-induced scratching was attenuated in a mouse model, and that mast cell degranulation was inhibited both in mouse skin in vivo and in human mast cells in vitro. We confirmed that the effect was due to ssON inhibiting the MRGPRX2 receptor, but also witnessed that ssON did not inhibit MRGPRX2 activation by all agonists tested, probably due to the nucleotide interacting with specific binding sites on the receptor, or having specific downstream signaling targets. Currently, we are looking at

48 the effects of ssON applied topically and if it might be beneficial in chronic itch conditions, using a mouse model for AD, Nc/Nga mice. If ssON proves useful in these experiments, it points to a potential and novel treatment option in chronic itch conditions associated with MRGPRX2 activation. Our analyses have shown that both mouse mast cell proteases mMCP6 and CPA3 play protective roles in itch induced by the pruritogens ET-1 and compound 48/80. ET-1 activates nerves directly and has the capability to degranulate mast cells, while compound 48/80 is a well established mast cell degranulator. Since CPA3 cleaves ET-1 directly in vivo we concluded that its protective effects were due to that cleavage. The role of CPA3 in compound 48/80- induced itch, and the part that mMCP6 plays in both types of itch remain spec- ulative and require further research. Mast cell degranulation and primary afferent activation release a variety of different mediators into the extracellular space, and downstream events can result in the release of more mediators still. We were able to confirm that ETAR activation was not a factor in compound 48/80-induced scratching in CPA3-deficient mice, and that the scratching phenotype of Mcpt6-/- mice when provoked with ET-1 was not dependent on CGRP but appears related to other factors besides direct ETAR activation. The next steps in elucidating these mechanisms could involve in vitro study of protease-deficient mouse mast cells to see how they respond to different pruritogens, if cleavage of certain substrates occurs and what the products are. It would also be informative to see if ET-1 does in fact cause mast cell degranulation in vivo in our itch model. In these papers we have only investigated acute itch but it would also be interesting to look at the effects of the mouse mast cell proteases in chronic itch conditions, such as in dry skin models. Taken together, the aim is that these studies will ultimately contribute to the understanding of the peripheral regulation of itch and pain and the many factors that come into play, such as immune cells, neuropeptides and their in- teraction in neurogenic inflammation. Hopefully, further elucidating the role of these different components will eventually help in the development of better treatments for burdensome conditions such as chronic pain and itch.

49 Acknowledgments

And the mercy seat is waiting And I think my head is burning And in a way I’m yearning To be done with all this measuring of truth. -Nick Cave & The Bad Seeds, Mercy Seat

I couldn’t help but laugh when I noticed the similarities in one of my favorite songs by Nick Cave and in what I felt at times, writing this thesis and thinking about the upcoming defense. Endless stream of scientific articles measuring truth (or some version of it) and in the end I will face a ceremony that hopefully will be less stressful than execution in the electric chair, like described in the song. But in a sense it will be an end of one type of life, the life of the PhD student, and the beginning of another.

This strange PhD life has been influenced and improved by so many wonderful people. First of all I would like to thank my excellent supervisor Malin Lagerström for her incredible support and mentoring. Thank you for knowing exactly when to push or encourage me and for always being available for ques- tions and discussions. Your work ethic and skills are an inspiration and I truly appreciate our collaboration.

I would also like to thank my co-supervisor Klas Kullander for providing support when Malin was not around and giving helpful feedback on those occa- sions when I have presented my research. Henrik Boije: thank you for helpful input when I was preparing lectures about the eye and vision, which is not exactly my field! I have had the good fortune to work with several amazing collaborators outside my department: Nick Spencer and his team at Flinders University in Australia, Gunnar Pejler and Mirjana Grujic at the Department for Medical Biochemistry and Microbiology here at Uppsala University, and Anna-Lena Spetz and Aleksandra Dondalska at Stockholm University. Thank you all so much, without you these projects would not have been possible. I also wish to acknowledge Lars Hellman at the Department of Cell and Molecular Biology at Uppsala University, who kindly shared new protease data with us.

50 I also wish to thank the skilled and helpful animal house staff both at BMC and SVA, and the wonderful admin ladies at the Neuroscience Department who have been very helpful and kind over the years. To my fellow members of the Sensory Circuits team, Marina, Fabio, Jon, Hannah (we will always have Venice), Haisha, Jonne (thanks for all your hard work for the whole unit!) and Akram (aka doorman): thank you all for your friendship, support, collaborations and helpful discussions through the years. I also wish to thank past members of the team, Tianle (who is a master in mouse tickling), Kasia (who took my under her wing when I was a master’s student and taught me most of the behavioral techniques I needed) and Bejan (thanks for all the support and friendship). I also wish to thank my project student Jessica Bergman for her itch scoring help in Paper IV. To all the other members of Developmental Genetics, both past and present: thank you all for making the unit an enjoyable work place, full of support, laughter and discussions. Try to keep the hood clean and the ethanol tanks full for once. So long and thanks for all the (zebra)fish. Special thanks to the wonderful Marina Franck and Remy Manuel, who kindly read the thesis and gave helpful feedback. To my best friends Svanhvít, Þura and Guðbjört: takk fyrir vinskapinn í gegnum árin þó að löndin hafi skilið okkur að. Þið eruð bestar! To my family: Elsku hjartans pabbi og mamma, takk fyrir allan stuðninginn og ástina í gegnum tíðina, þið bjugguð mér góðan grunn fyrir það sem hefur á eftir komið. Systkini mín, Guðný (sem málaði fallegu myndina á forsíðunni, hjartans þakkir), Herdís, Skúli og Marín: takk fyrir að vera þið, ég elska ykkur og vildi óska að ég gæti hitt ykkur oftar, þó að í mínum huga séuð þið enn fimm ára kríli með hor! Ég gæti síðan ekki verið heppnari með tengda- fjölskyldu, elsku Helga, Stulli, Guðrún, Stebbi og öll hin: takk fyrir allt gamalt og gott, og að láta mér líða eins og hluta af ykkar yndislegu, hressu fjölskyldu. Og takk öll fyrir að koma!

Og síðast en ekki síst, ástarþakkir til Steinars, Ylfu og Aríu. Meiri stuðning og ást er ekki hægt að biðja um en það sem þið hafið veitt mér. Það erfiðasta við þessa krefjandi vinnu hafa ekki verið langir og þreytandi dagar heldur fjar- veran frá ykkur. Á þeim stundum leið mér svolítið eins og Homer Simpson – Do it for her. Ég gerði þetta fyrir okkur öll. Ég vona að þið getið verið stolt af mömmu og við öll fagnað því að þessum áfanga sé loksins náð.

Ykkar einlæg/Yours sincerely, Elín Ingibjörg Magnúsdóttir

51 References

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71 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1596 Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine”.)

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