IL-17E exhibits pro-inflammatory effects in psoriasis through induction of innate immune responses and neutrophil recruitment

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Thesis

Reference

IL-17E exhibits pro-inflammatory effects in psoriasis through induction of innate immune responses and neutrophil recruitment

DA FONTE SENRA, Luisa Margarida

Abstract

Le psoriasis est une maladie inflammatoire chronique, affectant principalement la peau. De nombreuses études ont démontré le rôle clé de l'IL-17A dans cette maladie. Nous avons montré la surexpression d'IL-17E (une isoforme de la famille des IL-17) dans le psoriasis. In vitro, IL-17E induit la production par les macrophages de cytokines pro-inflammatoires et de chimiokines, particulièrement celles impliquées dans le recrutement de cellules immunitaires innées. In vivo, IL-17E induit une hyper prolifération des kératinocytes ainsi qu'une inflammation cutanée, caractérisées par la transcription de gènes impliqués dans des réponses immunitaires innées et dans le recrutement des neutrophiles. La délétion génétique d'IL-17E ou sa neutralisation améliore l'inflammation cutanée produite par l'application d'imiquimod ou par tape stripping. La neutralisation d'IL-17E particulièrement réduit le recrutement des cellules immunitaires innées telles que les neutrophiles et les macrophages.

Ces résultats montrent qu'IL-17E participe à l'inflammation cutanée en induisant des réponses immunitaires innées et [...]

DA FONTE SENRA, Luisa Margarida. IL-17E exhibits pro-inflammatory effects in

psoriasis through induction of innate immune responses and neutrophil recruitment. Thèse de doctorat : Univ. Genève, 2018, no. Sc. 5262

DOI : 10.13097/archive-ouverte/unige:111509 URN : urn:nbn:ch:unige-1115098

Available at:

http://archive-ouverte.unige.ch/unige:111509

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE

Département de Biologie Cellulaire FACULTÉ DES SCIENCES Professeur Jean-Claude Martinou

Département de Pathologie et Immunologie FACULTÉ DE MÉDECINE Professeur Wolf-Henning Boehncke

IL-17E exhibits pro-inflammatory effects in psoriasis through induction of innate immune responses and neutrophil recruitment

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Luísa Margarida da Fonte Senra de

Barcelos (Portugal)

Thèse n°5262

Genève

Centre d’Impression Uni-Mail 2018

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Acknowledgments

After four amazing years, I am now approaching the end of one of the most important stages of my professional life. Geneva has given me the opportunity to meet great people, and understand the pleasure of eating cheese fondue. Of course, this thesis would not have been possible without the help and support of many people, including my supervisor, colleagues, collaborators, friends and family. I would like to thank them all.

Firstly, I would like to thank the members of the jury, Prof. Jean-Claude Martinou and Prof.

Manfred Kopf for having kindly accepted to review and evaluate my thesis.

I would like to express my deepest gratitude to my thesis director, Prof. Henning Boehncke for accepting me in his lab and giving me the opportunity to work in exciting projects during these four years. I am grateful for his excellent mentoring, helpful advice and personal guidance during my PhD.

I would like to express my special thanks to my supervisor, Nicolo Brembilla, for his guidance, support and all scientific and non-scientific discussions that have definitely helped me to grow as a scientist.

I thank all past and present members of the lab: Craig, Ludovic, Julia, Bin, Romaine, Marylise and Sagar, for their advice and technical assistance. Thank you for your contribution throughout my project.

I also thank our collaborators in Lausanne, Dr. Curdin Conrad and Alessio Mylonas for their scientific and technical support with mice experiments.

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I would like to thank Prof. Cem Gabay and Prof. Carlo Chizzolini and respective lab members for their scientific input and technical help.

I thank my thesis godfathers Prof. Marc Chanson and Prof. Walter Reith.

I would like to thank all my friends from Geneva (Diana, Elsa, Montsé, Marta, Joana, Miguel, Gaia, Angela, Vasia, Cris) for all apéros, trips and amazing time spent together.

A special thanks to my beloved and lifelong friends Katia, Claudia and Ana Silvia (Bia) for their support and friendship.

I would like to thank my family for always being there for me, and their support even from far. Without them, this would not have been possible.

Finally, thanks to Diogo for keeping me happy and for giving me his support and comfort in difficult moments.

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Abstract

Psoriasis is a chronic recurrent immune-mediated and genetic skin disease. Despite the recent advances, the pathophysiology of psoriasis is still not fully understood. However, multiple pre-clinical, clinical and genetic studies have demonstrated the key role of IL-17A in psoriatic inflammation. IL-17A is the founding member of the IL-17 cytokine family, composed by five additional proteins (IL-17B to IL-17F). IL-17A signals via a heterodimeric receptor composed of IL-17RA and IL-17RC subunits. IL-17RA mediates the signaling not only of IL- 17A, but of other members of the IL-17 family, namely IL-17F, IL-17C and IL-17E.

Neutralization of either IL-17A or its receptor (IL-17RA) is so far the most effective therapeutic strategy in psoriasis.

We have initially analyzed the expression of IL-17 isoforms signaling via IL-17RA in psoriasis. We demonstrated that IL-17E is overexpressed in the lesional psoriatic skin.

Contrary to IL-17A, IL-17E was previously believed to be involved in the promotion and development of type 2 immune responses. Furthermore, it has been shown to dampen Th1 and Th17-driven inflammation by skewing the immune system towards Th2 immune responses. Thus, we aimed at investigating the role of IL-17E in psoriasis and more broadly in skin inflammation. First, we demonstrated that IL-17E expression is up-regulated in keratinocytes in the lesional skin of psoriatic patients, and that IL-17E targets dermal macrophages, inducing the production of pro-inflammatory cytokines and chemokines, particularly those involved in the recruitment of innate immune cells. Moreover, we observed that IL-17E expression in psoriasis positively correlates with the number of infiltrating neutrophils, but not T cells. These data suggested that IL-17E may play a pathogenic and pro- inflammatory role in psoriasis, particularly by promoting the recruitment of innate immune cells.

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We have further investigated the role of IL-17E by using mice models. In vivo, IL-17E induces keratinocyte hyperproliferation and skin inflammation, characterized by the transcription of genes involved in innate immune responses and the recruitment of neutrophils. Furthermore, we demonstrated that IL-17E is up-regulated during skin inflammation in mice. Genetic deletion or IL-17E neutralization ameliorates cutaneous inflammation driven by either imiquimod application or tape stripping. In particular, IL-17E neutralization reduces the recruitment of innate immune cells, such as neutrophils and macrophages, to the skin. We further demonstrated that IL-17E fails to induce the migration of neutrophils on its own, however, IL-17E-activated macrophages do promote the recruitment of neutrophils through secretion of IL-8. In addition to psoriasis, we show that IL- 17E is up-regulated in two other skin diseases (acute generalized exanthematous pustulosis and pyoderma gangrenosum) characterized by neutrophil-rich infiltrates.

Overall, these data suggest that IL-17E participates in skin inflammation by inducing innate immune responses and the recruitment of neutrophils. Therefore, we propose a new role for IL-17E, which is unrelated to the development of type 2 immune responses. This may lead to the identification of new therapeutic targets and the better understanding of the diverse effects of biologic therapies currently used to treat psoriasis, namely those inhibiting elements of the IL-17 axis.

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Résumé

Le psoriasis est une maladie inflammatoire chronique à médiation immunitaire et génétique, affectant principalement la peau. La physiopathologie du psoriasis est complexe et reste mal comprise. De nombreuses études précliniques, cliniques et génétiques ont démontré le rôle clé de l’IL-17A dans cette maladie. IL-17A est la première cytokine décrite de la famille des IL- 17 qui comprend 5 autres cytokines (IL-17B à IL-17F). IL-17A se lie à un récepteur formé de 2 sous-unités IL-17RA et IL-17RC.La sous-unité 17RA intervient également dans la signalisation d’autres membres de la famille IL-17, tels que IL-17F, IL-17C et IL-17E. La neutralisation de l’IL-17A ou de la sous-unité IL-17RA de son récepteur est actuellement la stratégie thérapeutique la plus efficace dans le traitement du psoriasis.

Nous avons débuté notre étude par l’analyse de l’expression des isoformes d’IL-17 signalant via la sous-unité IL-17RA du récepteur et nous avons démontré la surexpression d’IL-17E dans le psoriasis. Contrairement à IL-17A, IL-17E avait été préalablement décrite comme étant impliquée dans la promotion et le développement de réponses immunitaires de type 2, IL-17E pouvant atténuer l’inflammation provoquée par les Th1 et Th17 en orientant le système immunitaire vers les réponses immunitaires de type Th2. Pour tester ce concept nous avons investigué le rôle d’IL-17E dans le psoriasis et plus largement dans l’inflammation cutanée. Nous avons montré la surexpression d’IL-17E dans les kératinocytes des lésions psoriasiques. Cette augmentation d’IL-17E induit la production par les macrophages présents dans le derme de cytokines pro-inflammatoires et des chimiokines, particulièrement celles impliquées dans le recrutement de cellules immunitaires innées. Nous avons également observé une corrélation positive de l’expression d’IL-17E avec le nombre de neutrophiles infiltrants, mais pas avec le nombre de lymphocytes T. Ces résultats suggèrent un rôle pathogène et pro-inflammatoire d’IL-17E dans le psoriasis, en favorisant notamment le recrutement de cellules immunitaires innées.

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Ensuite nous avons étudié le rôle d’IL-17E dans des modèles de souris. In vivo IL-17E induit une hyper prolifération des kératinocytes ainsi qu’une inflammation cutanée, caractérisées par la transcription de gènes impliqués dans des réponses immunitaires innées et dans le recrutement des neutrophiles. De plus, nous avons montré une régulation positive d’IL-17E dans l’inflammation cutanée chez la souris : la délétion génétique d’IL-17E ou sa neutralisation améliore l’inflammation cutanée produite soit par l’application d’imiquimod soit par décapage (tape stripping). La neutralisation d’IL-17E particulièrement, réduit le recrutement des cellules immunitaires innées telles que les neutrophiles et les macrophages.

Nous avons démontré également que la cytokine IL-17E seule n’arrivait pas à induire la migration des neutrophiles. Ce sont les macrophages activés par IL-17E qui favorisent le recrutement des neutrophiles en sécrétant de l’IL-8. En plus du psoriasis, nous avons montré qu’IL-17E est régulée positivement dans deux autres maladies de peau caractérisées par des infiltrats riches en neutrophiles : la pustulose exanthématique aiguë généralisée et la pyoderma gangrenosum.

Globalement, ces résultats suggèrent qu’IL-17E participe à l’inflammation cutanée en induisant des réponses immunitaires innées et en favorisant le recrutement des neutrophiles.

Par conséquent, nous proposons un nouveau rôle pour IL-17E, non lié au développement des réponses immunitaires de type 2. Cette étude pourrait donc permettre d’identifier de nouvelles cibles thérapeutiques et d’améliorer la compréhension des divers effets des thérapies biologiques utilisées actuellement dans le traitement du psoriasis, notamment celles qui inhibent l’axe IL-17.

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List of abbreviations

ADAMTSL5 - ADAMTS-like protein 5

AGEP - acute generalized exanthematous pustulosis AMP - antimicrobial peptides

AP-1 - activator protein 1 APC - antigen presenting cell AS - ankylosing spondylitis

cAMP - cyclic adenosine monophosphate

CARD14 - caspase recruitment domain-containing protein 14 CCL - C-C motif chemokine ligand

CCR - C-C motif chemokine receptor CD - cluster of differentiation

CDKAL1 - CDK5 regulatory subunit associated protein 1 like 1 CDSN - corneodesmosin

CIA - collagen-induced arthritis CLA - cutaneous leucocyte antigen

CMC - chronic mucocutaneous candidiasis

CTLA - cytotoxic T lymphocyte associated protein CXCL - C-X-C motif chemokine ligand

CXCR - C-X-C motif chemokine receptor DC - dendritic cells

DNA - deoxyribonucleic acid

EAE - experimental autoimmune encephalomyelitis ERAP1 - endoplasmic reticulum aminopeptidase 1

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G-CSF - granulocyte colony-stimulating factor

GM-CSF - granulocyte-macrophage colony-stimulating factor GWAS - genome-wide association studies

HCR - coiled-coil helical rod protein HIV - human immunodeficiency virus HLA - human leucocyte antigen Hur - human antigen R

IBD - inflammatory bowel disease Ig - immunoglobulin

IL - interleukin

IL36RN - interleukin 36 receptor antagonist ILC - innate lymphoid cells

ILC2 - type 2 innate lymphoid cells IMQ - imiquimod

INF - interferons INF-a - interferon alfa INF-g - interferon gamma

iNKT - invariant natural killer T cell iNOS - inducible nitric oxide synthase JAK - janus kinase

JNK - JUN N-terminal kinase LCE - late cornified envelope

LCE3B - late cornified envelope protein 3B LCE3C - late cornified envelope protein 3C mAb - monoclonal antibody

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MAPK - mitogen-activated protein kinase mDC - myeloid dendritic cells

MHC - major histocompatibility complex

MICA - MHC class I polypeptide-related sequence A MS - multiple sclerosis

NCD - non-communicable disease NET - neutrophil extracellular traps NF-kB - nuclear factor kB

NFKBIA - NF-kB inhibitor alpha NK - natural killer cell

NK T - natural killer T cell NO - nitric oxide

PASI - psoriasis area severity index pDC - plasmacytoid dendritic cells PsA - psoriatic arthritis

PSOR - psoriasis susceptibility locus PSOR1 - psoriasis susceptibility locus 1 PSOR4 - psoriasis susceptibility locus 4 RA - rheumatoid arthritis

RAGE - receptor for advanced glycosylation end products RANKL - receptor activator of NF-kΒ ligand

RORg - RAR-related orphan receptor gamma ROS - reactive oxygen species

SEFIR - similar expression to fibroblast growth factor genes and IL-17R SF2 - mRNA splicing factor 2

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SLE - systemic lupus erythematosus SNP - single nucleotide polymorphisms

STAT3 - signal transducers and activator of transcription 3 Tc - cytotoxic T cell

TCR - T cell receptor

TGF-b - transforming growth factor beta Th - T helper cell

TIP-DC - TNF-a/ iNOS producing DC TIR - Toll/IL-1R

TLR - toll like receptor

TNF-a - tumor necrosis factor alfa

TNFAIP3 - tumor necrosis factor alfa induced protein 3 TNIP1 - TNFAIP3 interacting protein 1

TRAF - TNF receptor associated factor TRAF3IP2 - TRAF3 interacting protein 2 TYK2 - tyrosine kinase 2

VEGF - vascular endothelial growth factor VLA-1 - very-late antigen 1

WHO - World Health Organization ZNF 750 - zinc finger protein 750

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I. Introduction

1. Psoriasis

Psoriasis is a chronic recurrent, immune-mediated skin disease; patients often exhibit substantial comorbidity. It is associated with a great physical, psychological and social burden. Due to the visible disfiguration, disability and marked loss of productivity, it severely impacts the patients’ quality of life. Individuals suffering from psoriasis frequently experience social exclusion and discrimination, leading to increased rates of depression. Disease burden is further increased by several comorbid diseases including metabolic syndrome, cardiovascular diseases, non-alcoholic fatty liver disease, Crohn’s disease, and lymphoma [1].

In 2014, the World Health Organization (WHO) raised awareness of psoriasis as major global health problem, recognizing psoriasis as a serious non-communicable disease (NCD) [2].

The estimated prevalence of psoriasis in Europe and North America is around 2% [1].

Prevalence rates vary between geographic region, age and races, but are usually independent of the gender. Psoriasis tend to be more frequent at higher latitudes and in the Caucasian population [3,4]. These differences are particularly evident in the United States, where the prevalence among black population (0.45 to 0.7%) is significantly lower than in the remaining population (1.4 to 4.6%) [5]. Psoriasis can occur at any age, though prevalence increases over the lifecourse from 0.12% at age 1 year to 1.2% at age of 18 years [6,7].

1.1 Clinical and histological features

Psoriasis vulgaris, or chronic plaque psoriasis, is the most common clinical form of the disease and affects approximately 85 to 90% of the patients [8]. The disease presents as monomorphic and sharply demarcated erythematous plaques covered with silvery scales,

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lumbar regions. Common reported symptoms include pain, itching and bleeding from the skin patches.

Figure 1. Clinical manifestations of psoriasis.

Typical erythematous plaques with silvery scales (A) can be scattered (B), cover larger areas (C) or affect the entire body surface in erythrodermic psoriasis (D). Scalp involvement might be accompanied by non-scarring alopecia (E). Psoriatic arthritis (PsA) affects up to 30% of all patients (F). Nail changes are frequent and range from pitting and yellow or brown discolouration (G) to complete dystrophy (H). Psoriasis inversa occurs in intertriginous areas and is usually devoid of scales (I). Pustular psoriasis might occur in a generalised form (J, K) or localised (L, palmoplantar type and M). In children, the onset as guttate psoriasis might follow streptococcal infection of the upper respiratory tract (N) and affect any site of the body (O,P,Q) (Figure adapted from [1]).

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Four other clinical types of psoriasis have been reported: guttate or eruptive psoriasis, which is characterized by scaly droplet-shaped lesions; inverse psoriasis, also called intertriginous or flexural psoriasis, typically found in skin folds; pustular psoriasis, which can either take the form of palmoplantar pustulosis (pustular psoriasis of the palms and soles), or generalized pustular psoriasis, which is a rare and severe form of the disease; and erythrodermic psoriasis, a rare but life-threatening disease that affects the entire body surface and can arise from any form of psoriasis [1]. A considerable subset of psoriatic patients develop PsA, a potentially debilitating joint disease (Figure 1) [9].

Histological hallmark features of the psoriatic plaque include epidermal acanthosis (epidermal thickening) due to keratinocyte hyperproliferation; premature maturation and incomplete cornification of keratinocytes resulting in hyperkeratosis (thickened cornified layer) and parakeratosis (retention of nuclei in the stratum corneum), and marked elongation of the rete ridges (downward projections of epidermis between the dermal papillae).

The mitotic rate of basal keratinocytes is increased more than 50 times in psoriasis, so epidermal turnover takes 3 to 5 days instead of 28 to 30 days in healthy subjects [9]. In the dermis, a prominent inflammatory infiltrate mainly composed of T cells, dendritic cells (DC), macrophages and neutrophils, are present. Neutrophils and T cells, in less extent, also infiltrate within the epidermis. Neutrophils can either accumulate in the spinous layer of the epidermis forming pustules of Kogoj or in the stratum corneum, forming the so-called Munro’s microabcesses. The psoriatic microvasculature is characterized by dilated and contorted blood vessels, reaching the dermal papillar regions beneath the epidermis, which facilitate leucocyte migration into the skin. Characteristic vascular changes are thought to be mediated through angiogenic factors overexpressed in psoriasis and endothelial cell activation (Figure 2) [1].

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Figure 2. Histopathological features of psoriasis.

The psoriatic plaque is characterized by marked epidermal acanthosis, hyperkeratosis, and elongation of rete ridges (A, normal skin and B, lesional psoriatic skin; haematoxylin and eosin staining). Dilated and contorted dermal blood vessels reach into the tips of the dermal papillae (B, arrows). A mixed inflammatory infiltrate with neutrophils accumulate within the epidermis (B, asterisk). Immunohistochemical detection of CD3 reveals many T cells in the dermis and epidermis of lesional psoriatic skin (D, arrows), by contrast with normal skin (C).

Parakeratosis is also characteristic for lesional psoriatic skin (D, asterisk) (Figure adapted from [1]).

1.2 Comorbidities

Increasing epidemiologic and scientific evidence has led to the recognition that psoriasis is not limited to the skin, but should be regarded as a chronic systemic inflammatory disorder that is associated with or might even cause important comorbidities, such as inflammatory bowel disease (IBD), multiple sclerosis (MS) and rheumatoid arthritis (RA) [8]. The first observation of comorbid disease among patients with psoriasis was made in 1897, and

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reported an association between psoriasis and diabetes [10]. In 1961, postmortem examinations revealed a high prevalence of heart disease including coronary thrombosis and myocardial infarction in PsA patients [11]. Cumulating evidence has demonstrated that psoriasis is associated with numerous comorbid diseases, including metabolic syndrome, depression, Crohn’s disease, cancer, non-alcoholic fatty liver disease and cardiovascular diseases [10]. It is still controversial whether PsA, which has several features in common with psoriasis, should be considered an extracutaneous manifestation of psoriasis or a distinct disease with a distinct therapeutic spectrum, and therefore a psoriasis comorbidity [12].

Comorbid diseases substantially contribute to morbidity and mortality in patients with psoriasis, and are hypothesized to be the result of systemic inflammatory state associated with the skin disease [13,14]. Indeed, inflammation biomarkers are elevated in the blood of psoriatic subjects, and signs of inflammation can be readily detected by imaging techniques not only in the skin, but also in sites outside the skin [15,16]. Another possibility is that associations of comorbid diseases with psoriasis could be due to overlapping genetic basis of these diseases. For instance, is still unclear whether lymphoma is related to the pathophysiological mechanisms of psoriasis, or to its treatment [17]. In the next sections, some of the most common or clinically relevant comorbidities of psoriasis will be discussed in further detail.

1.2.1 Psoriatic arthritis (PsA)

PsA is a heterogeneous inflammatory arthropathy characterized by stiffness, pain and tenderness of the joins and surrounding ligaments and tendons, presented by a subset of psoriasis patients. In addition to peripheral joint involvement, PsA patients present other extra-articular manifestations of systemic inflammation [18]. PsA is a debilitating disease which increases the disease burden associated with psoriasis by further impairing quality of

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life and increasing cardiovascular risk [19]. It affects 6 to 42% of patients with psoriasis, depending on the definitions used and populations studied [10]. Prevalence might be even higher, given that PsA is considered an underdiagnosed disease. The prevalence of PsA increases with greater severity and duration of psoriasis [20]. Nevertheless, the severity of skin disease is only weakly associated with severity of joint disease. In most patients, PsA develops 10 years, on average, after the presentation of skin symptoms, but a small subset of patients with PsA develop symptoms of arthritis prior to the cutaneous disease [21].

Therefore, early detection is essential as early treatment improves disease outcomes.

Psoriasis and PsA have been assumed to result from similar genetic polymorphisms.

However, recent studies have found considerable genetic differences between psoriasis patients who develop PsA and those who remain free of joint involvement. In this regard, the heritability of PsA is 3-5 times higher than psoriasis [22,23]. There are also differences in the inflammatory microenvironment in the skin and in the synovial tissue. This likely explains why standard therapies for psoriasis may not be equally efficacious for PsA and vice versa [21].

1.2.2 Cardiovascular diseases

Among the comorbidities in psoriasis, cardiovascular diseases are of emerging significance, as they often affect patients’ mortality [24]. While patients with mild forms of psoriasis seem to not be at an increased risk, moderate and severe forms of the disease are associated with an increased risk for myocardial infarction and mortality [25]. Even though the association between severe psoriasis and increased cardiovascular risk is widely accepted, this association does not provide evidence for causality, and therefore the causes and consequences are still a question of intense debate. Several arguments favor the hypothesis that psoriasis is not an independent risk factor for cardiovascular diseases. Among them, it is the observation that

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psoriasis is associated with several diseases representing major cardiovascular risk factors such as diabetes mellitus, obesity, hypertension, dyslipidemia and metabolic syndrome, with the later characterized by the clustering of obesity, hypertension, insulin resistance and dyslipidemia [26]. This association has been proposed to result from common genetic susceptibilities shared between psoriasis and the referred comorbidities. Nonetheless, increasing evidence supports a direct link between psoriasis and cardiovascular diseases, favoring psoriasis as an independent risk factor. For instance, after adjusting for major cardiovascular risk factors (e.g. hypertension, diabetes and hyperlipidemia) mild psoriatic patients were shown to have a slightly elevated relative risk for cardiovascular events, while elevated relative risk was only found in patients with severe psoriasis [13]. Two meta-analysis have also shown that the cardiovascular risk correlates with the severity of the disease, suggesting a strong dose-response [27,28]. Moreover, longer duration of psoriasis is associated with increased cardiovascular risk [29,30]. Furthermore, most of genome-wide associations studies (GWAS) have failed to show a genetic association between psoriasis, metabolic syndrome and coronary artery disease [31]. Common pathophysiological pathways involved in either psoriatic and atherosclerotic plaque formation, which include among others Th1 and Th17-mediated inflammation, monocyte and neutrophil modulation, increased oxidative stress and angiogenesis, may also explain the increased cardiovascular risk in psoriatic patients. The pathogenic link between psoriasis and cardiovascular disease is likely to be provided by the so-called “psoriatic march” [32,33]. As observed in RA, a state of systemic inflammation is involved in psoriasis, evidenced by elevated circulating factors indicative of systemic inflammation and endothelial activation, such as C-reactive protein and platelet activation marker P-selectin [15,34–37]. According to this concept, systemic inflammation induces insulin resistance in endothelial cells, possibly via pro-inflammatory cytokines, which ultimately leads to endothelial dysfunction. Insulin resistance and

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endothelial dysfunction are key events in the development of atherosclerotic plaques [1].

Effective psoriasis therapies were shown to reduce the circulating levels of several pro- inflammatory cytokines and biomarkers of cardiovascular risk [38,39]. Therefore, the concept of “psoriatic march” provides the framework to explain how psoriatic inflammation drives cardiovascular comorbidity, via the development of atherosclerosis, independently from the presence of additional cardiovascular risk factors.

1.3 Etiology

The etiology of psoriasis is very complex and still not fully elucidated. However, it is widely accepted that psoriasis is influenced by both genetic and environmental risk factors.

Environmental triggers that are responsible for acute inflammatory processes in healthy individuals may be improperly controlled in genetically susceptible subjects, leading eventually to chronic inflammation and manifestation of the disease. Therefore, genetic factors seem to play a major role in the control and resolution of inflammation driven by environmental triggers. Genetic and environmental factors contributing to psoriasis will be discussed in the following sections.

1.3.1 Environmental factors

Cutaneous physical trauma (e.g. scratching, insect bites, tattoos), sunburns or chemical irritants are known to trigger the development of psoriatic lesions in previously uninvolved skin of psoriatic patients [40]. This response, known as Koebner phenomenon, occurs probably through the release of pro-inflammatory cytokines, stress proteins, adhesion molecules or auto-antigens. In addition, drugs such as beta-blockers, lithium, chloroquine and non-steroidal anti-inflammatory agents have been associated with induction or exacerbation of the disease [41]. However, the underlying molecular mechanisms are still not fully

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understood. Infections, particularly streptococcal infection of the upper respiratory tract, are strongly associated with the onset of guttate psoriasis in children [42]. Superantigenic T cell activation by streptococcal toxins, followed by an antigen-specific T cell response are thought to be responsible for the appearance of the skin lesions [43]. Human immunodeficiency virus (HIV) infections are also associated with psoriasis, since the prevalence of psoriasis in HIV- infected population is higher than the general population, and psoriasis seems to worsen in HIV patients [44]. On the other hand, other environmental factors, such as sun exposure, rather improve psoriatic symptoms in many patients.

1.3.2 Genetic factors

Epidemiological studies have clearly showed that the incidence of psoriasis among first and second-degree relatives is higher than in the general population [45]. Furthermore, the risk to develop psoriasis in monozygotic twins is up to three-fold higher as compared to dizygotic twins [45].

Linkage studies have identified multiple psoriasis susceptibility locus (PSOR), but most of the genes responsible for susceptibility are still not known [46]. PSOR1 is thought to be the major genetic determinant of psoriasis, accounting for 35 to 50% of the cases [7,47]. PSOR1 is located on the chromosome 6p within the class I region of Major Histocompatibility Complex (MHC). HLA-Cw6 was identified as the most likely susceptibility allele at PSOR1, given its larger and stronger association with psoriasis [47–49]. In addition, also the endoplasmic reticulum aminopeptidase 1 (ERAP1), which plays a role in processing peptides for loading into MHC class I molecules, has been associated with psoriasis, further supporting the role of T cells in the pathogenesis of psoriasis [50,51]. Although more involved in innate immune responses, sequence variants for MICA (MHC class I polypeptide-related sequence A), which is involved in activation of NK, NK T but also T cells, is also associated with psoriasis [52].

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In the past decades, GWAS have identified several novel susceptibility loci associated with psoriasis, outside of the MHC region (Table 1). Most of the candidate genes identified encode proteins involved in the regulation of both adaptive and innate immune responses; thus, linking the genetics of psoriasis with important pathogenic pathways of the disease.

Susceptibility loci included genes involved in the nuclear factor kB (NFkB) signaling pathway, such as TNFAIP3, TNIP1, NFKBIA and REL [50,53,54]. The NF-kB is a key transcription factor involved in cellular responses to stress and infections, and plays an important role in inflammation and apoptosis. Furthermore, mutations and single nucleotide polymorphisms (SNP) in the Caspase Recruitment Domain-Containing Protein 14 (CARD14), a gene involved in inflammasome and NF-kB activation, have been found in patients with severe forms of psoriasis [55–57]. GWAS have also implicated several components of the IL-23/IL-17 signaling pathway in the development of psoriasis. Sequence variants in the genes encoding interleukin-23 A (IL23A), interleukin-23 receptor (IL23R) and in the untranslated region of IL12B (p40) have been associated with increased psoriasis risk, supporting a role for Th17 cells in the pathogenesis of psoriasis [58–61]. IL23R variants have been also associated with other immune-mediated disorders, such as PsA and ankylosing spondylitis [62,63]. In addition, TRAF3IP2, which encodes for Act1 an adaptor protein involved in IL-17 and NF-kB signaling, and TYK2, a tyrosine kinase involved in the IL-23 signaling, were also identified as candidate psoriasis susceptibility genes [50,64]. Mutations in the signal transducer and activator 3 (STAT3), a key molecule mediating downstream signaling of several cytokines including IL-23, is also associated with psoriasis [65].

Other predisposing genes for psoriasis seem to be involved in epidermal differentiation/skin barrier rather than the immune system. Mutations in the zinc finger protein 750 (ZNF750), a protein normally expressed in keratinocytes, have been found in patients with autosomal- dominant psoriasiform inflammation [66]. Variants in the genes LCE3B and LCE3C,

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members of the late cornified envelope (LCE) gene cluster (PSOR4 locus), have also been strongly associated with increased risk of psoriasis [61,67,68].

Table 1. Major candidate susceptibility genes associated with psoriasis.

Gene/ Locus Pathway Other disease association References

PSOR1 (HLA-Cw6) Antigen presentation PsA [7,47,69]

ERAP1 Antigen presentation AS [50,51]

IL23A IL-23/IL-17 PsA [54]

IL12B IL-23/IL-17 PsA, IBD, AS, MS [54,58,60,61]

IL23R IL-23/IL-17 PsA, IBD, AS [54,58,59,63]

TYK2 IL-23/IL-17 IBD, SLE [50]

STAT3 IL-23/IL-17 IBD, MS [65]

TRAF3IP2 IL-17/NF-kB PsA [50,64,70]

REL NF-kB PsA, RA [50,71]

NFKBIA NF-kB PsA [50,53]

TNFAIP3 NF-kB PsA, SLE, RA [50,54]

TNIP1 NF-kB PsA, SLE [50,54]

CARD14 NF-kB PsA [55–57]

Abbreviations: AS, ankylosing spondylitis; IBD, inflammatory bowel disease (including Crohn’s disease and ulcerative colitis); MS, multiple sclerosis; PsA, psoriatic arthritis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus.

Some susceptibility genes were also identified in other diseases. For instance, variants in the CDKAL1 gene, have been associated with psoriasis, as well as, type II diabetes and Crohn’s disease, supporting the existence of shared genetic determinants between psoriasis and some comorbid diseases [72,73]. Most of the candidate susceptibility genes implicated in psoriasis were also found to be strongly associated with PsA (e.g. IL12B, TRAF3IP2, TNIP1 and REL)

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[70,74]. Nevertheless, severe generalized pustular psoriasis has been independently and strongly associated with mutations leading to loss of function in the Interleukin-36 Receptor Antagonist (IL36RN) gene [75].

1.4 Pathogenesis

The understanding of the cellular and molecular mechanisms behind psoriasis has dramatically evolved over the last years. Until the 1970s, psoriasis was regarded as a skin disease solely driven by keratinocyte dysregulation and hyperproliferation [76]. Accumulation of immune cells within the psoriatic plaques was observed, but not considered to participate in the pathogenesis, and rather just a consequence of hyperproliferating keratinocytes. Over the next decades, the role of the immune system, and particularly T helper (Th) cells, was subject of intense investigation. Evolving concepts in the pathogenesis of psoriasis culminated in the current hypothesis where Th17 cells and IL-17A, the principal effector cytokine of Th17 cells, are thought to be key proximal regulators of psoriatic inflammation.

1.4.1 Historical perspective: the involvement of immune cells

First studies suggesting the involvement of the immune system in the pathogenesis of psoriasis revealed an increased number of infiltrating immune cells, especially T cells and DC, in the lesional skin of psoriatic patients [77]. Additional compelling evidence of a dysregulated immune system emerged from the observation that T cell specific depleting agents and immunosuppressive drugs, such as cyclosporine, improved psoriasis clinical signs and symptoms [78,79]; reports that bone marrow transplantation could either cure psoriasis or transfer the disease from donor to recipient [80,81]; and that T cells from psoriatic patients can elicit the psoriatic lesions in animal models [82]. A pivotal study further demonstrated that specific depletion of lymphocytes, but not keratinocyte proliferation, improved disease

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symptoms [83]. Therefore, these studies provided the foundations to set up the general hypothesis of psoriasis as a T cell-mediated disease.

In 1998, IL-12, a cytokine produced by antigen-presenting cells (APCs) that promotes the differentiation of naive T cells into Th1 effector cells, was found to be increased in psoriatic lesions [84]. In addition, TNF-a and interferon-gamma (INF-g), cytokines involved in cellular responses against viral and bacterial infections, which are expressed by Th1 cells, were also found increased in psoriatic patients [85,86]. These findings prompted the concept that psoriasis was mediated by activated Th1 cells, with TNF-a and INF-g as key soluble mediators of psoriatic inflammation.

In 2000, a breakthrough in the field of immunology occurred with the identification of the novel p19 protein, a subunit of the IL-23 cytokine [87,88]. While IL-12 is composed of p35 and p40 subunits, IL-23 is formed by p19 and p40 subunits. Psoriasis was incorrectly characterized as a Th1-driven diseased based, at least in part, on the assessment of p40 levels within the psoriatic lesions [84]. Subsequent studies showed that both IL-23 p19 and p40 subunits were increased in psoriasis, whereas the level of IL-12-specific p35 subunit was not increased [89,90]. In 2003, IL-23 was shown to induce the production of IL-17 from activated T cells, and to be fundamental for the commitment and/or the expansion of the recently discovered IL-17-producing CD4⁺ cells (Th17 cells) [91]. Gene expression analysis revealed that Th17 cells have a completely distinct cytokine profile compared to Th1 cells: Th17 cells express high levels of IL-17A, as well as IL-17F, IL-22 and TNF-a. In addition, Th17 cells were shown to be involved in tissue inflammation and autoimmune responses (Figure 3).

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Figure 3. Cytokine environment regulates Th cell differentiation into functional subsets.

IL-4 drives polarization of naïve T cells into Th2 phenotype, while IL-12 (constituted by p40 and p35 subunits) and IL-23 (composed of p40 and p19 subunits) induce the differentiation into Th1 and Th17 subsets, respectively (Figure adapted from [76]).

The discovery of Th17 cells in the lesional skin of psoriasis lead to the reassessment of the Th1 cell hypothesis, as dominant mediators of tissue damage in psoriasis. In addition, GWAS have reproducibly identified several psoriasis susceptibility genes that are linked with components of the IL-23 and IL-17 signaling pathways, further focusing attention on Th17 cells in this disease. Overall, these findings laid the groundwork for characterizing psoriasis as a Th17-mediated disease, where Th1 cells rather play a secondary role.

1.4.2 Current concepts

The current view of the pathogenesis of psoriasis proposes that environmental factors, such as stress, drugs and physical trauma, trigger the initiation of psoriatic lesions in genetically predisposed individuals carrying the disease-associated susceptibility alleles [8]. In the

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initiation phase, stressed keratinocytes release pro-inflammatory mediators, including IL-1b, IL-6 and TNF-a, that activate resident myeloid DC (mDC). Additionally, keratinocytes can also release self-DNA that forms complexes with the antimicrobial peptide (AMP) LL-37, which in turn activate plasmacytoid dendritic cells (pDC). INF-a produced by pDC leads to activation of myeloid DC. Activated mDC migrate into the skin-draining lymph nodes to present antigens, and secrete mediators such as IL-12 and IL-23, driving the differentiation of naïve T cells into Th1 and Th17 effector cells, respectively. Effector cells recirculate via lymphatic and blood vessels infiltrating into the skin attracted by a chemokine gradient. Th1 cells expressing cutaneous leucocyte antigen (CLA) and CXCR3, and Th17 cells expressing CLA and CCR6 are attracted by the keratinocyte-derived chemokines CXCL9, CXCL10, CXCL11 and CCL20, which ultimately leads to the formation of the psoriatic plaque.

Inflammatory mediators secreted by Th17 cells (i.e. IL-17A, IL-17F and IL-22), Th1 cells (i.e. TNF-a, INF-g), and inflammatory DC (i.e. TNF-a, NO) act on keratinocytes leading to activation, proliferation and production of AMP, inflammatory cytokines and T cell and neutrophil attracting chemokines, thus creating a positive feedback loop in the inflammatory process responsible for plaque maintenance. Moreover, CD8 T cells migrate into the epidermis through interactions between a1b1 integrins (very-late antigen-1 (VLA1)) on T cells and collagen IV at the basement membrane, further contributing to the pathogenesis of the disease. Feedback loops involving keratinocytes, fibroblasts and endothelial cells lead to tissue reorganization and deposition of extracellular matrix (e.g. collagen and proteoglycans) (Figure 4).

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Figure 4. Immunopathogenesis of psoriasis.

Complex interactions between skin resident cells and cells of the immune system orchestrate the pathologic changes in pre-psoriatic skin. Key processes during disease initiation and maintenance include dermal DC activation and induced differentiation of naïve T cells into Th1 and Th17 through IL-12 and IL-23; secretion of inflammatory mediators (e.g. IL-17A, IL-17F, IL-22 and TNF) by pathogenic T cells in the skin; keratinocyte activation; further infiltration of immune cells, including T cells and innate immune cells such as neutrophils mediated by keratinocyte-derived chemokines. The latter cell types further amplify the inflammatory process through expression of soluble mediators (Figure adapted from [92]).

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Our understanding of the role of numerous immune cells and inflammatory mediators involved in the pathogenesis of psoriasis had continued to progress over the past years. In addition to T cells and DC, the role of innate immune cells, such as neutrophils, mast cells, macrophages, and skin resident cells, such as keratinocytes and endothelial cells, in the immunopathogenesis of psoriasis has been subject of intense investigation. In the next sections, the role of key cellular players in psoriasis will be discussed in further detail.

1.4.3 Role of DC and plausible autoantigens

DC are key sentinels of the immune system, acting as messengers between the innate and adaptive immune compartments. Skin DC can be classified according to their functional properties and localization in the skin. Langerhans cells are the main DC subset in the epidermis, residing in the suprabasal layers regularly spaced among keratinocytes, while mDC are found throughout the whole dermal compartment. In addition, different DC subtypes might have specific functional properties, such as secretion of pro-inflammatory mediators, by inflammatory DC or production of type I interferon by pDC [92].

Increasing evidence of a dysregulated cross talk between the innate and the adaptive immune system has emerged in psoriasis [93]. mDC are increased in psoriatic lesions [94] and become activated by cytokines including TNF-a, INF-a, INF-g and IL-1b, secreted by innate immune cells and skin resident cells. As stated above, activated mDC present antigens and secrete mediators such as IL-12 and IL-23, driving the activation, differentiation of Th1 and Th17 cell that infiltrate into the psoriatic lesions. DC also have strong pro-inflammatory capacity. A specific subset, commonly named TIP-DC, expressing TNF-a and inducible nitric oxide synthase (iNOS) is present within the lesions, and it has been proposed to play a role in the pathogenesis of psoriasis [95].

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Though the innate immune system has been proposed to drive chronic inflammation in psoriasis, the exact mechanisms just began to be deciphered. pDC, the main producers of type I interferons, are a unique DC subset with key effector functions on antiviral immune responses. pDC have been shown to infiltrate in early psoriatic lesions possibly triggering disease onset and/or exacerbation [96]. Activation of the intracellular Toll-like receptor 9 (TLR9) on pDC through complexes of self-DNA, released by stressed or dying cells, with the keratinocyte-derived AMP cathelicidin (CAMP)/LL-37 leads to INF-a production, followed by the activation of mDC and activation of adaptive immune responses [97]. Complexes of self-RNA with LL-37 have also been shown to activate pDC and mDC through activation of TLR7 and TLR8, respectively [98]. In addition, complexes of LL-37 with self-DNA were reported to activate other cell types, such as monocytes [99].

A key question that remains unresolved concerns the autoimmune nature of psoriasis and the contribution of possible autoantigens to the disease. A restricted usage of the T cell receptor repertoire has been reported in psoriasis, suggesting antigen-specific immune responses [100].

Furthermore, the strong association between streptococcal infection and psoriasis, and the sequence homology between streptococcal M peptides and keratins, such as keratin 17, supported the hypothesis that some keratinocytes proteins could function as autoantigens in psoriasis [43]. Nevertheless, formal proof for a role of an autoantigen in psoriasis was lacking. Only recently it has been proposed that the AMP LL-37 could function as an autoantigen in psoriasis [101]. Lande R. et al. have shown that LL-37 is recognized and able to activate CD4⁺ and/or CD8⁺ psoriatic T cells, inducing their proliferation. LL-37-specific T cells displayed effector functions, by producing IFN-γ and Th17 cytokines, key cytokines in the pathogenesis of psoriasis. However, only 46% of the patients analysed in the study harboured autoreactive T cells to LL-37 [101]. Therefore, this study provided initial evidence of the presence autoantigens in psoriasis, at least in a subset of patients. Furthermore, it

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uncovered a potential mechanism through which an endogenous AMP can function as T cell autoantigen, by breaking innate tolerance to self-DNA/RNA in psoriasis. LL-37 represents the first example of an AMP, which can stimulate both innate and adaptive immune cell compartments in autoimmune settings.

Another study has identified ADAMTS-like protein 5 (ADAMTSL5) an additional plausible autoantigen [102]. ADAMTSL5 is produced by melanocytes and induces a T cell response in HLA-C*06:02–restricted psoriatic CD8⁺ T cells. ADAMTSL5 stimulation of CD8⁺T cells from psoriatic patients induced the expression of IL-17A, a key effector cytokine in the pathogenesis, supporting the role of ADAMTSL5 as melanocyte autoantigen in psoriasis.

Furthermore, in the lesional skin of psoriatic patients numerous CD8⁺ T cells were found in close contact with melanocytes. These CD8⁺ T cells contained lytic granules polarized towards the melanocytes. Lytic granules are an important effector mechanism of CD8⁺ T cells, and suggest TCR-mediated activation.

In summary, DC play a key role in the pathogenesis of psoriasis by presenting putative autoantigens, such as LL-37 or ADAMTSL5, promoting the activation and differentiation of naïve T cells into Th17 and Th1 effector cells. So far, the relevance of autoantigens in psoriasis is still a controversial issue.

1.4.4 Role of T cells

The central role of T cells in the pathogenesis of psoriasis has been established based on several clinical observations and experimental findings using animal models. The successful treatment of psoriasis patients with immunosuppressive drugs, such as cyclosporine, which inhibits T cell proliferation and cytokine production was the first clinical evidence supporting a pathogenic role for T cells in psoriasis. Other T cell targeted therapies were also shown to

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improve disease signs and symptoms. In addition, several animal models of psoriasis have confirmed the importance of T cells during initiation and maintenance of the disease.

Skin resident T cells, rather than recruited T cells, are thought to have a major role in skin immune homeostasis and pathology. In psoriasis, skin-resident T cells were proposed to be sufficient to maintain the disease state during the chronic relapsing course of the disease, without the necessity to recruit circulating immune cells [103]. In fact, human healthy skin harbors a considerable population of skin-resident T cells (2 × 10¹⁰), which is more than twice the total number of T cells circulating in the blood, and most of CLA⁺ skin-homing lymphocytes reside in the skin under physiological conditions [104].

The key role of T cells in skin inflammation has been established in several mouse models.

Injection of activated autologous lymphocytes, especially T cells, in the dermis of symptomless skin from psoriatic patients engrafted into severe combined immunodeficient (SCID) mice has been shown to induce the development of psoriatic plaques [82].

Furthermore, uninvolved skin from psoriatic patients grafted into immunodeficient AGR mice (AGR mice is deficient for type I and type II INF receptors and Rag2, thus lacking T, B and NK cells) was shown to spontaneously develop typical psoriatic lesions, without injection of activated lymphocytes or other stimuli [105]. The development of psoriatic lesions suggests that skin-resident T cells could be essential and sufficient for the development of the disease.

Moreover, migration of pathogenic T cells into the epidermis, through interaction between VLA-1 and collagen IV on the basement membrane, was shown to be required for the development of psoriatic phenotype [106]. These observations were also confirmed in human.

Matos et al. demonstrated that clinically resolved psoriatic skin lesions contain residual oligoclonal populations of T cells, that produce IL-17A in both resolved and active lesions.

The putative pathogenic T cell clones were most frequent in resolved lesions, but also present in lower frequency in the non-lesional skin of the same patient. These cells preferentially

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utilize TCR Vβ repertoire and have unique psoriasis-specific TCR sequences, further suggesting a pathogenic role of skin resident T cells in disease initiation [107].

The role of pathogenic T cells, particularly of Th17 phenotype, has been show in a variety of chronic inflammatory diseases, including psoriasis. Th17 cells and their downstream effector cytokines (e.g. IL-17A, IL-17F, IL-22 and TNF-α) are increased in psoriatic skin and peripheral blood of patients, and act on keratinocytes and other skin resident cells, inducing keratinocyte proliferation and amplification of local inflammation [108–110]. Moreover, IL- 23, a key cytokine for the development and activation of Th17 cells, is increased in psoriatic lesions, mainly produced by DC and macrophages [90,111]. Th1 cells are also increased in the lesional skin of psoriatic patients [108,110]. It has been suggested that Th1 and Th17 cells may collaboratively contribute to autoimmune diseases, including psoriasis [112]. In addition to Th17 and Th1 cells, a distinct population of IL-22 producing CD4⁺ cells (Th22), was recently reported to be increased in psoriasis [108]. IL-22 induces keratinocyte proliferation and was shown to be required in a murine psoriasiform model, thus suggesting a pathogenic role of Th22 cells in psoriasis [113,114]. Overall, these pathogenic T cells are implicated in the pathogenesis of psoriasis, by interacting with other cell types, and particularly keratinocytes, thus creating a chronic inflammatory environment.

1.4.5 Role of keratinocytes

Despite the central role of immune cells, and particularly T cells, in the pathogenesis of psoriasis, there is increasing evidence suggesting the participation of keratinocytes in both initiation and maintenance of the disease through amplification of specific effector responses [115]. DC and Th17 cell-derived mediators, such as TNF-a, IL-17A and IL-22, activate epidermal keratinocytes inducing their proliferation and cytokine production. In turn, keratinocytes secrete high levels of pro-inflammatory cytokines (e.g. IL-1, IL-6 and TNF-a)

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[116] and chemokines, particularly the ones involved in neutrophil (e.g. CXCL8, CXCL1), monocyte (e.g. CCL2) and T cell recruitment (e.g. CXCL9, CXCL10, CXCL11 and CCL20), thus creating a positive feedback loop on the inflammatory infiltrate [92]. In addition, keratinocytes express a set of small proteins known as AMP, including LL-37, b-defensins, lipocalin, and the calcium-binding S100 protein family members S100A7 (psoriasin), S100A8 (calgranulin A) and S100A9 (calgranulin B), which may also activate innate immune receptors. Some of these S100 proteins are abundant in psoriasis and may play a major inflammatory role through activation of RAGE [117,118] and TLR receptors [119,120].

Furthermore, AMP have strong chemotactic capacity for multiple inflammatory cell types, thus further amplifying inflammation [118,121].

The concept that dysregulated immune responses are responsible for the initiation of psoriatic lesions was challenged by reports suggesting that epidermal alterations are sufficient to initiate skin lesions. For example, abrogation of JunB/activator protein 1 (AP-1) in keratinocytes triggers chemokine/cytokine expression, which promotes neutrophil and macrophage recruitment, culminating in a phenotype histologically resembling psoriasis [122]. AP-1 is a transcription factor involved in the regulation of cell proliferation, differentiation and stress responses in several tissues [123]. Furthermore, constitutive activation of STAT3, which is downstream cytokine receptors such as IL-23R, in keratinocytes, leads to psoriasiform inflammation, thus suggesting that alteration in key signaling pathways on keratinocytes can alter skin homeostasis and induce pathology [124].

Whether keratinocytes or lymphocytes are the primers of psoriasis is still not known.

Importantly, keratinocytes must be considered an integral part of the skin immune surveillance system given their role in pathogen recognition, production of inflammatory mediators and functional development of T cells [125].

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1.4.6 Role of innate immune cells

Contrary to the intense research focus on adaptive immune cells in psoriasis, the contributions of innate immune cells, such as neutrophils and macrophages, to the disease have been much less appreciated.

Neutrophils migrate early into developing lesions and accumulate in the dermis and epidermis, a hallmark feature of psoriatic plaques. These cells constitute the first line of immune defense and secrete important inflammatory mediators such as reactive oxygen species (ROS), hydrolytic enzymes and multiple cytokines (e.g. IL-1a, IL-1b, IL-6, TNF-a, IL-23) which induce proliferation, alter differentiation, and activation of keratinocytes and immune cells [126,127]. Moreover, neutrophils can release AMP in a process called NETosis.

Neutrophil extracellular traps (NETs) decorated with AMP, such as LL-37, and IL-17A have been recently shown in the skin of psoriatic patients, suggesting an additional role for neutrophils in the pathogenesis of psoriasis [128]. On the other hand, upon activation by inflammatory mediators, skin resident and immune cells secrete neutrophil attracting chemokines (e.g. IL-8, CXCL1) which further activate and recruit neutrophils into the lesions, thus creating a positive feedback loop [115,129]. Clinical targeting of neutrophils, through neutralization of IL-8 with a specific monoclonal antibody, has not proven to be sufficiently effective in psoriasis. The limited success of this approach is explained most likely by a redundant effect, since several mediators share overlapping and mutually compensating functions in leucocyte recruitment [130]. Nevertheless, neutrophil depletion ameliorates psoriasiform lesions in mice [131]. Moreover, neutrophil depletion by granulocyte apheresis is particularly effective in patients with generalized pustular psoriasis, a subset characterized by prominent neutrophilic infiltration [132,133].

Recently, the role of innate immune cells in the pathogenesis of psoriasis has been highlighted. Cumulating evidence suggests that most of IL-17A secreted during inflammation

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is produced by innate immune cells, such as neutrophils and mast cells, rather than Th17 cells [128,134]. Therefore, this hypothesis brings innate immune cells, particularly neutrophils, to the centre of IL-17-dependent pathophysiology of psoriasis. In this regard, neutrophils were shown to be an early target of IL-17-neutralizing therapies [135]. Significant clinical responses were associated with an early and extensive clearance of cutaneous neutrophils parallel to the normalization of keratinocyte abnormalities and reduction of IL-17-inducible neutrophil chemoattractants (e.g. CXCL1, CXCL8), whereas the effect on T cell and DC was substantially delayed. This study provided evidence for a new model in the pathogenesis of psoriasis, where a neutrophil-keratinocyte crosstalk, in which IL-17 (potentially derived from neutrophils), stimulates keratinocyte production of chemokines that, in turn promote further influx of neutrophils into psoriatic lesions [135]. IL-17 neutralization is thus associated with an early interruption of the crosstalk between innate immune cells and resident skin cells, while full and long-term clinical response is associated with disruption of adaptive immune responses.

Macrophages are also increased in the lesions of psoriatic patients, particularly around the epidermal-dermal junction, but their role in the pathogenesis of the disease is still not fully understood [136–140]. These cells are important mediators in the modulation of innate immune responses, and produce large amounts of inflammatory cytokines such as IL-6, IL-1b and TNF-a [141]. Even though psoriasis is considered a T cell mediated disease, macrophages were reported to play a pathogenic role, at least in some mouse models of psoriasiform skin inflammation. These studies have shown that TNF-a, produced mainly by macrophages, drives skin inflammation [142–144]. TNF-α is a critical cytokine in the pathogenesis of psoriasis, and other chronic inflammatory diseases, as shown by the remarkable clinical success of TNF-α–neutralizing agents, in the treatment of psoriasis [145]. Furthermore, regulatory T cells were shown to counteract macrophage-mediated skin

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inflammation [146,147]. The factors that lead to macrophage infiltration and activation in the skin remain to be addressed, but may be dependent on auto-reactive T cells and/or aberrant epidermal signaling [144]. Interestingly, neutrophils do not contribute to skin inflammation in those models [141,143]. Considering the increasing evidence that macrophages may be relevant for the pathogenesis of psoriasis, it is fundamental to address those findings in human or xenogenic models.

2. IL-17 cytokine family

The IL-17 family is composed by six structurally related cytokines: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (also called IL-25) and IL-17F. IL-17 family members are highly conserved across evolution and homologues have been found in several species [148–150]. Among all the members, the biological function and regulation of IL-17A and IL-17F are by far the best characterized. These two cytokines are also the closest related members, sharing 55%

homology at the amino acid level, and are often co-expressed as disulfide-bond homodimers or heterodimers [151]. IL-17B, IL-17D and IL-17C amino acid sequences overlap 29%, 25%

and 23%, respectively, with IL-17A. IL-17E is the most divergent member of the family and shares only 16% sequence homology [152,153]. IL-17 family members mediate their biological function via receptors expressed at the surface of target cells. Similar to the IL-17 family, the IL-17 receptor (IL-17R) family is composed by five receptor subunits: IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17RE, identified largely based on their amino acid sequence homology with IL-17RA, the founding member of the IL-17R family. IL-17 cytokines signal through a heterodimeric receptor composed by a different combination of IL- 17R subunits [154,155]. IL-17A and IL-17F bind to the same receptor complex composed of IL-17RA and IL-17RC subunits [156]. IL-17RA is also a common subunit of the receptor complex for IL-17E (which is composed by IL-17RA and IL-17RB subunits) [157], and IL-

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17C (composed by IL-17RA and IL-17RE subunits) [158]. Current knowledge on IL-17B and IL-17D signaling is limited, but IL-17B is believed to signal through IL-17RB. Whether the receptors for IL-17B and IL-17D are heterodimeric, and whether IL-17RA is a common co- receptor is still not known (Figure 5).

Figure 5. IL-17 cytokine and receptor family.

IL-17A is the prototypical cytokine of the IL-17 family that includes five other members (IL- 17B–F). Cytokines function as homodimers, with IL-17A/F also forming heterodimers. Each cytokine uses a specific receptor complex composed of the IL-17 receptor subunits (IL- 17RA–E). IL-17A, A/F and F dimers bind to IL-17RA–RC, IL-17C binds to the IL-17RA–RE complex, IL-17E interacts with the IL-17RA–RB. Although IL-17B can associate with IL- 17RB biochemically, the function of this interaction is unclear. Likewise, it is unknown whether IL-17B also associates with IL-17RA. The receptor for IL-17D is unknown, and IL- 17RD is an orphan receptor. Abbreviations: CBAD, C/EBPβ activation domain; SEFEX, SEFIR extension; SEFIR, similar expression of fibroblast growth factor and IL-17Rs (Figure adapted from [159]).

Due to its unique structural features, IL-17R family was not recognized to be related to any previously known class of cytokine receptors. Moreover, this family mediates signaling events that are clearly distinct from other cytokines, particularly those involved in adaptive immunity. Cytokines involved in Th1 and Th2 immune responses typically trigger JAK- STAT signaling pathways, while IL-17 family members signal through an Act1 adaptor

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protein, culminating in the activation of several pro-inflammatory factors associated with innate immune responses [154]. Members of the IL-17R family are single transmembrane receptors that contain a conserved cytoplasmic SEFIR (similar expression of fibroblast growth factor and IL-17R) domain, that is related to the Toll/IL-1R (TIR) domain found in the IL-1 and TLR family [160,161]. Upon ligand binding to the IL-17R complex, the SEFIR domain engages with Act1 (also known as CISK) and leads to the activation of downstream pathways. The recruitment of Act1 is a hallmark feature of the IL-17A signaling and its required for all known IL-17A-mediated effects [162,163]. In addition to its role as an adaptor protein, Act1 acts as a ubiquitin ligase which recruits and ubiquitinates TRAF6 inducing a cascade of molecular events, leading to the phosphorylation and consequent proteasomal degradation of IkB, allowing nuclear translocation of NF-kB and consequent transcription of NF-kB targeted-genes [164–166]. TRAF6 has also been linked with the activation of mitogen-activated protein kinase (MAPK) pathways, including the extracellular signal- regulated kinase (ERK), p38, and JUN N-terminal kinase (JNK) [159]. However, the dominance of these pathways in response to IL-17A seems to be cell-specific. Furthermore, Act1 can recruit TRAF2 and TRAF5 proteins, inducing the sequestration of RNA decay factors (e.g. mRNA splicing regulatory factor 2, SF2) [167] and activation RNA-binding proteins (e.g. human antigen R, HuR) [168], which result in increased mRNA stability of the target genes (Figure 6).

Therefore, in addition to upregulation of inflammatory genes via de novo transcription, IL-17 members can promote the expression of a large number of genes by controlling mRNA stability. Indeed, stabilization of target mRNA transcripts seems to be one of the mechanisms by which IL-17A additively and synergistically interact with other pro-inflammatory cytokines [169].

IL-17E exhibits pro-inflammatory effects in psoriasis through induction of innate immune responses and neutrophil recruitment

Thesis

Reference

IL-17E exhibits pro-inflammatory effects in psoriasis through induction of innate immune responses and neutrophil recruitment

IL-17E exhibits pro-inflammatory effects in psoriasis through induction of innate immune responses and neutrophil recruitment

Table of Contents

Acknowledgments

Abstract

Résumé

List of abbreviations

I. Introduction