The different dimensions of how inflammation impacts cutaneous biology

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Thesis

Reference

The different dimensions of how inflammation impacts cutaneous biology

BREMBILLA, Nicolo

Abstract

In this thesis, I will discuss some of my recent articles that clarify the role of T helper (Th)17 and Th22 lymphocytes in two diseases presenting, among others, a state of chronic skin inflammation: systemic sclerosis (SSc) and psoriasis. SSc is characterized by an uncontrolled fibroblast response leading to dermal fibrosis. Psoriasis is characterized by an abnormal response of keratinocytes, leading to epidermal modifications. I will first refer to our discovery of the aryl hydrocarbon receptor as a priming factor of human skin-homing T cells expressing IL-22 (Th22 cells). I will next introduce the dermal dimension of Th22/Th17-mediated cutaneous inflammation, showing how these cells participate to the inflammatory component of SSc while rather restraining fibrosis. Finally, I will discuss the epidermal dimension of IL-17-mediated inflammation in psoriasis, showing that IL-17E, an IL-17A isoform, favors the recruitment of neutrophils in the skin. This will lead to the discussion of future research, including the use of human skin reconstituted models to study the epidermal-dermal communication during cutaneous [...]

BREMBILLA, Nicolo. The different dimensions of how inflammation impacts cutaneous biology. Thèse de privat-docent : Univ. Genève, 2018

DOI : 10.13097/archive-ouverte/unige:112256

Available at:

http://archive-ouverte.unige.ch/unige:112256

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

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Clinical Medicine Section

Department of internal medicine specialties

The different dimensions of how inflammation impacts cutaneous biology

Thesis submitted to the Faculty of Medicine of the University of Geneva

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for the degree of Privat-Docent by

Nicolò Costantino BREMBILLA

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Geneva 2018

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ABBREVIATIONS used in the text

ADAMTSL5: ADAMTS-like protein 5 AhR: aryl hydrocarbon receptor BP: binding protein

CCL: Chemokine (C-C motif) ligand CCR: C-C chemokine receptor CD: cluster of differentiation

C/EBP: CCAAT-enhancer-binding proteins CLA: cutaneous lymphocyte antigen CXCL: chemokine (C-X-C motif) ligand 1 DC: dendritic cell

DTH: delayed type hypersensitivity

EAE: experimental-induced autoimmune encephalomyelitis ECM: extra-cellular matrix

ERK: extracellular signal-regulated kinase FC: fragment crystallizable

FITC: Fluorescein isothiocyanate FN: fibronectin III-like domain

G-CSF: Granulocyte-colony stimulating factor

GM-CSF: Granulocyte-macrophage colony-stimulating factor HLA: Human Leukocyte Antigen

IFN: interferon Ig: immunoglobulin IL: interleukin

ILC: innate lymphoid cell

IMID: immune-mediated inflammatory disease JAK: janus kinase

JNK: c-Jun N-terminal kinase LC: Langerhans cell

LTi: lymphoid tissue inducer

MADISH: Metabolising Aquired Dioxin Induced Skin Hamartomas MAPK: mitogen-activated protein kinase

MCP: Monocyte Chemoattractant Protein MMP: Matrix metalloproteinase

NF-kB: nuclear factor kappa-light-chain-enhancer of activated B cells NK: natural killer

PG: prostaglandin

PRR: pattern recognition receptor RIG: retinoic acid-inducible gene-I-like RORγt: RAR-related orphan receptor gamma

SEFIR: similar expression of fibroblast growth factor genes and I-17Rs SSc: systemic sclerosis

STAT: Signal transducer and activator of transcription TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin

TCR: T cell receptor

TGF: transforming growth factor Th: T helper

TNF: tumor necrosis factor Treg: T regulatory cell

WHO: World Health Organization

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1. SUMMARY

Inflammation is a physiological response aiming at restoring the lost homeostasis of a tissue.

In case the initial inflammatory trigger cannot be cleared, or in presence of immunological abnormalities, inflammation may turns into a chronic state leading to pathology. In this thesis, I will discuss two distinct diseases that manifest, among others, in a profound inflammatory- dependent alteration of the skin: systemic sclerosis and psoriasis. The former is characterized by an uncontrolled fibroblast responses leading to dermal fibrosis. The latter is characterized by an abnormal response of keratinocytes, leading to epidermal modifications. I will thus discuss a selection of my recent articles that clarify the roles of the recently discovered Th22 and Th17 cells and related cytokines in the establishment of the dermal and epidermal manifestations that characterize systemic sclerosis and psoriasis, respectively.

I will first refer to our discovery of the aryl hydrocarbon receptor as a factor involved in the priming in vivo of human skin-homing T cells that express IL-22 in absence of other T cell lineage-defining cytokines, now known as Th22 cells.

I will next present the dermal dimension of Th22 and Th17-mediated cutaneous inflammation. In that regard, I will provide a few articles demonstrating that these cells are characteristically augmented in systemic sclerosis, and that clinical management of digital ulcers with prostacyclin analogs, often associated with amelioration of skin symptoms, results in the in vivo expansion of the Th17 subtype. This will lead to core findings underlying that Th22 and Th17 cells participate to the inflammatory component of systemic sclerosis while having no effect on, or rather restraining, dermal fibrosis.

Finally, I will discuss the most recent part of my research, which addresses the epidermal dimension of IL-17 mediated inflammation using psoriasis as model disease. I will show that IL-17E, an isoform of IL-17A, leads to the establishment of a pathologic cross-talk between keratinocytes and macrophages, leading to the recruitment of neutrophils into the skin. This latter work represents the basis of redefining the role of IL-17E in skin inflammation.

Based in these findings, I will briefly discuss the future direction of my research which will encompass the use of human skin reconstituted models to further study the communication between the epidermal and dermal compartments in skin inflammation, particularly in the context of Th17 cells and cytokines related to the IL-17 family.

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2. INTRODUCTION

The following paragraphs aim at introducing the scientific background of the articles described in this thesis, which collectively provide new insights on how T cell-dependent cutaneous inflammation impacts the biology of skin resident cells. The introduction is organized in two sections. The first, section 2.1, will guide through the main inflammatory cells, namely Th17 and Th22 cells, and related cytokines studied in this thesis. The second, section 2.2, will provide a general overview of the architecture of the skin and will describe the main physiopathological events taking place in systemic sclerosis (SSc) and psoriasis, which were used as model disease in the presented articles.

2.1. The inflammatory reaction

The term “inflammation” comes from the Latin root inflammare (in + flamma), literarily meaning: “to set on fire”. This definition reflects particularly well the overall process. As early as in 30 BC, the Roman encyclopaedist Celsus first described in Book III of his treatise

“De Medicina” the cardinal signs of inflammation: “rubor et tumour cum calore et dolore”

(redness and swelling, with heat and pain). Several centuries later, in mid-1800, the German pathologist Rudolph Virchow saw inflammation as inherently detrimental and proposed

“functio laesa” (loss of function) as the fifth cardinal sign[1]. Today, we know that inflammation is an innate immune reaction that occurs in response to several types of insults (infectious agents, chemical compounds, physical injuries, immune reactions, etc), which aims at eradicating the causing agent to re-establish the homeostasis of the affected tissue[2].

Thus, resident sentinel cells, such as dendritic cells (DCs) and macrophages, recognize a noxious stimulus via pattern recognition receptors (PRR), resulting in the release of several mediators that cooperatively coordinate the immune response. Vasoactive amines, arachidonic acid metabolites, cytokines and kinins are among the most important mediators produced at the site of inflammation. The blood flow locally increases (heat and redness), the vasculature becomes permeable leading to extravasation of fluids and leucocytes (swelling and pain), and the causing agent is ultimately cleared by the action of infiltrated phagocytes, mainly neutrophils. The innate part of inflammation also sets the base for the activation of antigen-specific adaptive responses through migration of activated DCs to secondary lymphoid organs, where the priming of naïve B and T cells is achieved. Newly generated antigen-specific effector T leucocytes travel back to the inflamed tissue, where they help innate mechanisms to clear the causing agent. At later stages of the inflammatory process, an immunological memory arises allowing an immediate, stronger and more specific immune response to antigen re-exposure. Inflammation is also a means for repair, since the causing agent itself and the immune response to it inevitably cause tissue damage. The clearance of

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5 infiltrated phagocytes by macrophages and their reprogramming towards a pro-resolving phenotype characterizes later phases of inflammation. Repair is achieved by shifting the pattern of mediators produced from pro-inflammatory molecules to anti-inflammatory mediators and growth factors. Together, these molecules allow the termination of the inflammatory response while promoting tissue regeneration[3].

The above-described scenario refers to a self-limiting acute inflammation. When the causing agent persists, cannot be cleared, or when abnormal immune reactions take place, like in autoimmunity or in allergic reactions, inflammation may turn to a chronic state with serious detrimental effects for the host. In this situation, inflammation, tissue injury and attempts at repair coexist in varying combination, leading to diverse clinical manifestations. While neutrophils are the main immune cell infiltrating the tissue during acute inflammatory reactions, chronic responses are dominated by the recruitment of several different types of innate and adaptive leukocytes. T cells may be the dominant immune population seen in autoimmune and hypersensitivity reactions[4]. These cells greatly amplify the inflammatory reaction and shape the nature of the immune response thought release of specific soluble mediators, such as cytokines. Uncontrolled inflammation, sometimes with a chronic recurrent course, is a core component of a broad group of disorders categorized under the name of immune-mediated inflammatory diseases (IMIDs). The pathogenesis of several such disorders is often linked to the presence of an unbalanced cytokine network resulting from the dominance of a particular T cell subset. In the following chapter, I will provide an overview of how helper T cell responses participates in the inflammatory response with a particular focus on the role of Th17 and Th22 cells, topic of this thesis.

2.1.1. CD4 T cells in inflammation.

CD4 T lymphocytes, also named T helper (Th) cells, actively participate to the inflammatory response, especially in chronic conditions. In 1986, Mosmann and Coffman initially proposed a Th1-Th2 dichotomy to explain cell- and antibody-mediated immune responses, respectively[5, 6]. On one hand, Th1 cells, via production of IFNγ and TNFα, stimulate macrophages, elicit a delayed-type hypersensitivity reaction (DTH) and drive immunity towards intracellular microorganisms. On the other hand, Th2 cells, by secreting IL-4, IL-5 and IL-10, participate in antibody production (in particular IgE) and eosinophilic inflammation, and are critical for immunity to extracellular pathogens and helminths.

Abnormal activation of Th1 cells is linked with autoimmunity, while exaggerated Th2 cell responses are responsible for asthma and allergic inflammatory diseases. Since these seminal works, several additional subsets have been added to the list, including Th17, Th22, Th9 and

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6 regulatory T cells (Treg) (Figure 1). Treg cells were reported to suppress effector cells, inflammation and autoimmunity[7, 8]. The balance between these subsets in the tissue is a key aspect determining the outcome of the inflammatory and immune responses. As discussed in the following chapters, Th17 and Th22 cells are particularly relevant in inflammatory and autoimmune conditions, and exert their effects mainly in peripheral tissues, such as the skin.

Figure 1. Polarized CD4+ T cell subsets. (Adapted from [9] Chizzolini and Del Galdo.

EULAR On-line Course on Systemic Sclerosis; MODULE 2: Pathogenesis: Immunological changes. 2011)

2.1.2. Th17 cells

In 2003, Aggarwal and colleagues reported that the cytokine IL-23 promoted the production of IL-17A by activated T cells[10]. A couple of years later, in 2005, the lack of IL-17A- producing cells in IL-23^-/- mice was linked to autoimmune resistance in a model of experimental-induced autoimmune encephalomyelitis (EAE)[11]. On the basis of the characteristic cytokine profile, these IL-17A-producing cells were named Th17 cells[12].

Th17 cells develop in vitro from naïve CD4 precursors upon TCR stimulation in presence of IL-1β, IL-6, and IL-23. TGFβ has also been proposed as a triggering factor for Th17 cells, although its role is more debated. Th17 cells characteristically express the transcription factor RORγt and express the skin-homing receptor CCR6 and CD161 at their surface. Th17 cells can broadly be subdivided into two populations: protective Th17 cells expressing IL-17A and

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7 IL-10; and pathogenic Th17 cells expressing IL-17A in conjunction with other pro- inflammatory molecules, such as IL-22 and GM-CSF. Several studies support the notion that IL-23 is essential to drive pathogenicity [13].

At difference to Th1 and Th2 cells, Th17 cells retain a high degree of plasticity and are epigenetically poised to co-express different lineage-specific transcription factors (such as T- bet and GATA3, master transcription factors of Th1 and Th2 cells, respectively) and effector cytokines. Such plasticity endows Th17 cells with an enhanced ability to traffic to several anatomically different sites and promotes an array of diverse functions[14]. Experiments in IL-17A and IL-17 receptor-deficient mice demonstrated that Th17 cells provide a host defense against diverse pathogens, including bacteria, parasites, fungi, and viruses[15], whereas deficiency in IL-17 signaling is particularly linked with Candida albicans infection in humans[16]. Moreover, Th17 cells have been implicated in the onset/progression of several human inflammatory and autoimmune disorders, including psoriasis, multiple sclerosis, ankylosing spondylitis, as well as their respective murine models[17]. Our group, together with others, has reported that increased frequency of Th17 cells also characterized systemic scelorisis[18-23]. My contribution in that regard will be discussed in section 3.2 (reference [21]) and 3.3 (reference [18]) of this thesis. The unique features of the cytokine IL-17A, which will be discussed in the following chapter, mediate the main effects of Th17 cells.

IL-17A

IL-17A, the main product of Th17 cells, was cloned in 1993 from a rodent activated T cell hybridoma and shows an atypical sequence for a cytokine, being highly homologue to the T- cell tropic Herpesvirus Samirii[24, 25]. In addition to Th17 cells, other innate and adaptive immune cells have been reported to produce IL-17A, including neutrophils, mast cells, group 3 innate lymphoid cells (ILC), NK cells, CD8 T cells and γδ T cells[13]. IL-17A signals via a receptor composed of two subunits: IL-17RA and IL-17RC. Upon ligand binding, Act1 is recruited to the receptor, leading to the activation of distal signaling events, including STAT3, C/EBP, MAPK and NFκB pathways [26, 27].

IL-17A mainly participates in protective immunity at boundary tissues, including the skin, gut and lung. IL-17A mediates its immunological functions by inducing pro-inflammatory cytokines, chemokines, anti-apoptotic factors and anti-microbial molecules in non- hematopoietic cells, such as epithelial cells. The factors induced by IL-17A are poised towards the activation of a neutrophil/Th17 cell-dependent immune reaction. This includes IL-8, a potent neutrophil chemoattractant, G-CSF, a survival factor for neutrophils and

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8 CCL20, which favors Th17 cell recruitment. In addition, IL-17A directly contributes to leucocyte migration and tissue remodeling by promoting the secretion of metalloproteases, and support cell homeostasis. Of note, IL-17A not only induce de novo transcription of key pro-inflammatory molecules, but also acts by stabilizing target mRNA. In that regard, IL-17A synergize with and potentiate the effects of many other inflammatory mediators, among which type-I cytokines are the most relevant [28-30].

The IL-17 family of cytokines

In early 2000, genomic sequencing identified several proteins structurally related to IL-17A:

IL-17B, IL-17C, IL-17D, IL-17E (also called IL-25) and IL-17F (Figure 2). Together, these cytokines form the IL-17 family. IL-17A and IL-17F share the highest homology (55%), while IL-17E appears to be the most divergent member of the family. The IL-17 cytokines exert their functions as homodimers, with the exception of IL-17A and IL-17F that can also associate as heterodimer. All members of the family signal via a heterodimeric receptor composed by a different combination among 5 distinct receptor subunits: IL17RA to IL17RE.

Knowledge on the signalling pathways induced by the receptor subunits other than IL-17RA (briefly described in the above paragraph) is sparse. Despite that, IL-17RA is shared by several isoforms (i.e. IL-17A, IL-17F, IL-17C and IL-17E), thus it is likely that multiple cytokine of the family use similar signalling mechanisms [26, 27].

Consistent with that, many IL-17 isoforms share similar biological functions. This is particularly true in the case of IL-17F and IL-17C. IL-17F is often co-expressed with IL-17A, being IL-17A and IL-17F found in the same gene locus[31]. IL-17F induces generally weaker responses than IL-17A, whereas it might have a particular important role in joint disease.

Along this line, dual IL-17F/IL-17A inhibition seems to have a better therapeutic efficacy than neutralization of IL-17A alone in psoriatic arthritis patients[32]. At difference to IL-17A and IL-17F, IL-17C is mainly produced by epithelial cells rather than immune cells. IL-17C is linked to skin inflammation in mice, as its overexpression is sufficient to trigger a psoriasiform phenotype, whereas its ablation protects from imiquimod-induced effects[33, 34]. The knowledge of the functions of IL-17B and IL-17D remains instead limited. Overall, these isoforms promote pro-inflammatory responses in non-immune cells and appear to regulate tumor and joint immunity.

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9 Figure 2. Schematic representation of the different IL-17 cytokines and their receptors. The main signalling events downstream the activation of the IL-17RA are summarized. FN:

Fibronectin III-like domain. SEFIR: Similar Expression of Fibroblast growth factor genes and IL-17Rs domain. (Brembilla NC et al., The IL-17 family of cytokines in Psoriasis. Front Immunol 2018 (under review))

IL-17E, also referred to as IL-25, shares only 16% homology with IL-17A and is the most divergent cytokine of the family. Several immune cell types, as well as epithelial cells, are capable of IL-17E production. Unlike the other members of the family, IL-17E has been implicated in type 2 responses and was shown to counteract Th17 responses during systemic autoimmune inflammation, such as in EAE and rheumatoid arthritis[35, 36]. Transgenic overexpression of IL-17E or its systemic administration leads to the production of Th2 cytokines, eosinophilia, ad epithelial cell hyperplasia in the gut and the lung. Thus, IL-17E was found to favour immune protection against helminth infections and contribute to allergic inflammation[37, 38]. Contrary to its relatively longstanding role in systemic inflammatory diseases, IL-17E has only been recently implicated in skin inflammation. We and others have highlighted that IL-17E may amplify the innate inflammatory responses specifically in the skin independently of type 2 responses[39-41]. My contribution to the identification of the effects of IL-17E in skin inflammation will be further discussed is section 3.5 (reference [40]).

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10 2.1.3. Th22 cells

In fall 2009, several distinct groups identified the existence in humans of a subset of CD4 T cells capable of IL-22 but not IL-17 and IFN-γ production, named Th22 cells. Th22 cells, whose existence in mice remains still debated, express the skin-homing receptor CCR4, CCR6, CCR10 and CLA, and are consistently thought to exert their functions in skin, where they participate to immunosurveillance [42-44]. IL-6 and TNF are the major cytokines driving Th22 polarization in humans [42, 45, 46]. Of interest, TGFβ, which is required for optimal Th17 differentiation in the mouse, inhibits IL-22 production. Th22 cells do not express Th1, Th2 or Th17 lineage-associated transcription factors, although RORγt may promote IL-22 production in some circumstances. The differentiation of Th22 cells requires instead the transcription factor aryl hydrocarbon receptor (AhR), whose ligands include both environmental toxins and endogenous breakdown products of the aromatic amino acids[44, 47-49].

Th22 cells were shown to contribute to host defence against microbial pathogens and to promote tissue repair. Th22 cells have been identified in several inflammatory skin diseases, including psoriasis and atopic dermatitis, and autoimmune disorders presenting skin manifestation, including SSC[22, 50]. My contribution to the definition of AhR as the master regulator of Th22 cells (section 3.1, reference [47]), as well as of Th22 as part of the pathogenesis of SSc (section 3.2, references [21, 51]) will be discussed later in this thesis.

The following paragraph will describe the role of IL-22 in skin diseases, as main of the Th22 effects are mediated by this cytokine.

IL-22

IL-22 belongs to the IL-10 family of cytokines, including IL-19, IL-20 IL-24 and IL-26. IL- 22 was originally described as a Th1-associated cytokine and only later was associated to Th17 and Th22 cells. In addition to these cells, IL-22 is produced by different lymphoid populations, including CD8+ T, classical and non classical (NK-22) NK cells, NKT cells, γ/δ T, DC and lymphoid tissue inducer (LTi) cells[52]. IL-22 signals via a receptor consisting of IL-22R1 and IL-10R2 subunits. The latter also function as an accessory receptor chain for the IL-10, IL-26 and IL-28/IL-29 receptor complex. In addition to the cell surface receptor complex, IL-22 may bind to a soluble, single chain IL-22 receptor named IL-22 binding protein (IL-22BP). IL-22 induces rapid activation of JAK1/Tyk2 signaling, leading to phosphorylation of STAT1, STAT3 and STAT5. IL-22 also activates three major MAPK pathways: ERK, JNK and p38 [53].

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11 IL-22 does not serve the communication between immune cells since cells of the hematopoietic origin do not express IL-22R1. It mainly acts on epithelial cells of the digestive tract, respiratory system and the skin, where it promotes antimicrobial defence, protection against damage, epithelial homeostasis and regeneration. Similar to IL-17A, IL-22 also supports the migration of immune cells and the remodelling of the inflamed tissues by favouring the secretion of metalloproteases. In addition, IL-22 favours induction of pro- inflammatory mediators, mainly chemoattractant for T cells (CXCL9, CXCL10, CCL2, CCL20), although requires the synergistic activity of TNF for mounting an efficient response.

Finally, IL-22 has profound effects on proliferation and differentiation of epithelial cells through the activation of the STAT3 pathway. Given its action on tissue homeostasis and survival, IL-22 is a key cytokine in the wound healing process[28, 54].

Depending on the target tissue and experimental condition, IL-22 may promote either protective or pathogenic immune responses. IL-22 is expressed in several chronic inflammatory and autoimmune conditions, including psoriasis, atopic dermatitis, lupus erythematosus, allergic asthma, rheumatoid arthritis and systemic sclerosis[51, 55]. Despite that, the precise roles played by IL-22 in these diseases remain often unclear and need further investigation. My contribution to the identification of the function exerted by IL-22 in Systemic Sclerosis will be discussed in section 3.4 (reference [51]).

2.2. Cutaneous biology

The skin is one of the largest organs in the human body and represents the most exposed surface to the environment. The skin is however not a simple mechanical barrier, but a dynamic tissue ensuring an effective communication with the external environment. The skin protects from water loss, helps to maintain body temperature, functions as sensory tissue, and allows synthesis of vitamins and hormones. In addition, it protects the interior tissues from a wide range of exogenous factors, such as pathogens, ultraviolet radiation and physical/chemical irritants. To perform such actions, the skin evolved as a complex organized tissue, which relies on sophisticated and well-coordinated interactions between skin stromal cells (i.e keratinocytes and fibroblasts) and immune cells. Several immune cells patrol the skin at steady state, and many more cells are efficiently and rapidly recruited in case of inflammation. A versatile network of lymphatic vessels, ensuring appropriate communication with skin-draining lymph nodes, also participates in efficient immune protection. The resulting equilibrium between the skin immune systems and the colonizing cutaneous microbiome guarantee the skin hemostasis. When the skin barrier is disrupted, or the immune surveillance is compromised, an inflammatory reaction takes place in order to re-establish the

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12 lost homeostasis. Cytokine and other inflammatory mediators may however profoundly affect the normal function of skin stromal cells, such as fibroblasts and keratinocytes. In presence of chronic cutaneous inflammations, these responses may ultimately lead to pathology. In the following chapters, I will provide an overview of the structure of the skin and the immune cells that populate the cutaneous environment. This will lead to introduce two inflammatory disorders, systemic sclerosis and psoriasis, whose clinical manifestations reflect an inflammatory-driven activation of fibroblasts and keratinocytes, respectively.

2.2.1. The structure of the skin

The skin is composed of an outer epidermis overlying an inner dermis, separated by a basement membrane (Figure 3). The epidermis is a multilayered stratified epithelium primarily constituted by keratinocytes. The epidermis comprises 4 distinct layers, named (from the bottom-most layer): basal, spinous, granular, and cornified layer [56]. The basal layer contains epidermal stem cells and proliferating keratinocytes. Pigmented melanocytes and nerve-ending cells essential for light-touch, known as Merkel cells, are also present in this layer. As basal keratinocytes differentiate and migrate upwards in the spinous layer, they lose the ability to undergo mitosis. Keratinocytes of this layer adopt a more polyhedral shape and start producing secretory organelles derived from the Golgi, called lamellar bodies.

Lamellar bodies contain lipid-derived molecules, enzymes including lipases and lipid hydrolases, and anti-microbial peptides[57]. Dendritic cells, known as Langerhans cells, provide immunosurveillance in this stratum. In the above granular layer, keratinocytes start to lose their nuclei and the cytoplasm organizes in keratohyalin granules, containing structural proteins such as pro-fillagrin, loricrin and involucrin. Moving upwards to the cornified layer, granular cells undergo terminal differentiation becoming anucleated dead, flattened cells called corneocytes. Keratohylin granules and lamellar bodies are released during terminal differentiation leading to the formation of a cornified envelope composed of structural proteins beneath the membrane surrounded by an impermeable, lipid-containing membrane.

Corneocytes are attached to each other through modified desmosomes, called corneodesmosomes, which are degraded in the uppermost layers to allow shedding[58]. In normal epidermis, the proliferation of basal keratinocytes is thus compensated by desquamation of cornified cells, leading to a complete renewal of the epidermis every 4-5 weeks[59].

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13 Figure 3. The skin. Schematic representation of the skin. The box shows details of the epidermal structure. (Adapted from [60] Marieb and Hoehn. Human Anatomy & Physiology, 8^th edition. Pearson International Edition 2010 and [61] S. Starndring. Gray’s Anatomy - The Anatomical Basis of Clinical Practise. 40^th edition. Churchill Livingstone 2008)

The dermis locates below the epidermis, and it is separated from it by a basement membrane enriched in type IV collagen. The dermis is a mesenchymal tissue composed by extra-cellular matrix (ECM) components, such as collagens (principally type I and type III collagen in human skin), proteoglycans and elastin. Fibroblasts populating the dermis are the cells responsible for the production and degradation of the surrounding ECM[62]. Two different dermal layers are recognized by histological assessment: the papillary and reticular dermis (Figure 3). The papillary dermis locates close to the basement membrane, shows a high density of fibroblasts and is composed of dense, thin, poorly oriented collagen bundles.

Nipple-like extensions of the dermis into the epidermis, called dermal papillae, are present in the uppermost part of the papillary dermis. Dermal papillae augment the surface area between the dermis and epidermis, resulting in improved exchanges of nutrients and other substances.

The beneath reticular dermis is characterized by low cell density and thick, directional- oriented collagen fibers. The ECM composition in these layers likely reflects differences in fibroblast identity, and specific fibroblast subsets have been reported to form dermal papillae and dermal sheath around hair follicles[63]. Under the reticular dermis lies the dermal white adipose tissue, also called hypodermis. Several appendage structures are found within the dermis[64]. These include hair follicles as well as sweet and sebaceous glands, which derived from a specialized invagination of the epithelium. The dermis also contains nerve sensors for touch and heat, and blood-lymphatic network providing nourishment and waste removal for both the dermis and the epidermis. Most of the immune cells residing in the skin at steady state are found in the dermis, as described in the following paragraph.

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14 2.2.2. Overview of cutaneous immunity

The epidermis forms a first physical barrier to external insults. In addition, epidermal keratinocytes are formidable sentinels for the frontline detection of pathogens, as they express multiple PRR, including Toll-like receptors, Nod-like receptors, RIG-I–like receptors and C- type lectins[65]. Furthermore, keratinocyte can sense a wide range of non-specific stimuli, such as UV radiation and chemicals. In response to pathogens or upon activation through cytokines, keratinocytes produce a range of pro-inflammatory cytokine (IL-1β, TNF, IL-6, etc), chemokines (IL-8, IP-10, etc) and growth factors (GM-CSF, etc), which are critical in the recruitment of immune effectors and in the regulation of the immune response. In addition, keratinocytes can directly neutralize an invading pathogen by producing antimicrobial-peptides (LL37, S100 proteins)[66].

Although keratinocytes play important innate immune functions, the epidermis is also enriched in immune cells specialized to the activation of adaptive responses: the Langerhans cells (LCs). LCs are a subset of dendritic cells located in the spinous layer that possesses dendrites extending through tight junctions to the cornified layer. LCs, observed for the first time over 150 years ago by Paul Langerhans, express several PRR, including C-type lectins, FC and C3 receptors. Upon activation, LCs migrates to the draining lymph node, where settle in the T cell zone to initiate an adaptive response[67].

Several types of immune cells locate in the dermis. Among the most important are DCs, macrophages, mast cells, ILCs and T cells. All cooperatively interact to mediate an adequate immune surveillance and efficient immune response. DCs represent another means by which the invading pathogen is seen and presented to T cells, upon antigen processing and migration to secondary lymphoid structure. Several dermal DC subsets have been described in human skin. Each subset has specific immune functions, while together guaranteeing an efficient immune responses to a variety of diverse pathogens[67, 68]. Besides DCs, resident- macrophages represent an additional important sensor of skin invading pathogens and damage. Skin macrophages do not have migratory capacities and have a poor antigen- presenting ability, whereas displaying enhanced scavenging properties. Several subsets of macrophages have also been reported, based on their ability to foster inflammation (M1) or rather dampen the immune response to promote tissue repairs (M2) [69].

The skin also represents a large reservoir of lymphocytes. It is estimated that the skin contains nearly twice as much T cells than the blood. Skin T cells essentially have a memory phenotype, and consist of both CD8 and CD4 expressing cells, with around 5-10% of the latter being Treg cells [70]. Part of the skin-homing T cells recirculates between the skin and

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15 the blood, whereas some reside in the tissue and are disconnected from the circulation. These latter cells are referred to as tissue-resident memory T cells (TRM). Recent studies revealed that T_RMcells rather than recruited T cells drive skin immunosurveillance. Episodes of recurrence in chronic skin inflammatory disorders may well be linked to the presence of these cells [71, 72].

Whether resident or re-circulating, T cells participate to the inflammatory response, in part by performing direct effector functions, in part by secreting cytokines and chemokines. The type of T cells activated and the soluble mediators produced are dependent on the encountered pathogen and ensure an effective pathogen-targeted immune response. As referenced to in section 2.1.1, Th1 cells via secretion of IFNγ arms macrophages and NK cells to control viral infections, Th2 cells neutralize extracellular parasite via IL-4-dependent IgE production, whereas Th17 cells stimulate host defense relying on IL-17A to protect against extracellular bacteria and fungi [7, 8]. In chronic inflammatory and autoimmune conditions, T cell responses may become predominant and the homeostasis of the tissue may be lost, leading to serious pathological manifestation. In the section below I will introduce two pathological conditions I have been working on in the last years: systemic sclerosis and psoriasis. These disorders represent an excellent example of how inflammatory cells affect the cutaneous biology: SSc with regard to dermal fibroblasts, psoriasis with regard to epidermal keratinocytes.

2.2.3. Systemic sclerosis and fibrosis

Fibrosis refers to the accumulation of ECM, especially collagen, in a given tissue. In most circumstances, fibrosis is a physiological self-limiting process aimed at repairing an injury.

Scar formation during wound healing is an example in that regard. When the process is not appropriately controlled and terminated, the excessive ECM deposition results in altered tissue and organ architecture, leading to dysfunction and pathology. Fibroblasts play key roles in fibrosis due to their ability to regulate the homeostasis of the ECM: they synthesize matrix components on one hand, and produce matrix-degrading enzymes, of which matrix metalloproteinases (MMP) are the most relevant, on the other hand. Excess of ECM synthesis over degradation takes place in pathological fibrosis [73].

Progressive fibrosis of the skin and internal organs, associated with diffuse fibroproliferative microangiopathy and inflammation, are characteristic traits of Systemic Sclerosis (SSc) [74]

(Figure 4A and B). SSc is a rare orphan disease of unknown etiology, which presents more commonly in Afro-Americans and shows a remarkable female predominance[75]. Abnormal immune-inflammatory events in response to environmental factors, such as virus infections

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16 and exposure to chemicals, are thought to play a role in disease pathogenesis in genetically predisposed individuals[76]. SSc has no cure and is associated with high mortality, making it a major challenge for physicians and patients. Clinical manifestations are heterogeneous, with a rate of progression that varies from a relatively stable to rapidly progressive disease. Two major clinical subsets are described according to the extent of cutaneous thickness. The limited form (lSSc) is defined by skin involvement distal to the elbows and knees, whereas patients affected by the diffuse form (dSSc) present extensive skin involvement[74].

Figure 4. Systemic Sclerosis. A) Raynaud phenomenon in a patient of SSc. B) Perivascular mononuclear cell inflammatory infiltrate in the dermis of SSc (*). An expansion of the collagenous reticular dermis (**), with loss of subcutaneous adipose tissue and encasement of sweat glands (***) is visible (hematoxylin-eosin). C) Scheme representing the role of Th1 and Th2 cell subsets in SSc (A & B - adapted from [77] Hochberg et la., Rheumatology, 6^th edition. Mosby 2014; C - adapted from [78]Brembilla and Chizzolini, T cell abnormalities in systemic sclerosis with a focus on Th17 cells. Eur Cytokine Netw. 2013:23(4):128-39).

Several lines of evidence suggest that immune and chronic inflammatory events are key to the initiation of the fibrotic process. The synthesis of collagen is maximal above a mononuclear cell inflammatory infiltrate, which characteristically precede the development of vascular and fibrotic alterations [79, 80]. In addition, infiltrating T cells express an activated phenotype and show a limited TCR usage, suggesting that they have undergone clonal expansion in response to a yet unidentified specific (auto-)antigen[81, 82]. In agreement with a possible autoimmune involvement, antinuclear antibodies are frequently observed early in the disease course[83]. Genetic evidence also supports a role for the immune system in the pathogenesis

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17 of SSc. Almost all genetic polymorphisms associated with SSc susceptibility maps to genes of the innate and adaptive immune system, with the HLA region being the most strongly associated[84]. Finally, immunosuppression is so far the only strategy that gives hope for a cure. Results from three clinical trials testing autologous stem cell transplantation indicate a dramatic and durable improvement in skin fibrosis, pulmonary functions and quality-of-life measures[85].

Soluble mediators produced by T cells could be of particular importance in driving collagen synthesis by fibroblasts[86]. Several evidence suggests the existence of a predominant Type 2 response in SSc skin, characterized by cytokines such as IL-4, IL-5 and IL-13. In line with this data, IL-4 producing Th2 cells and double positive CD4+CD8+ cells, as well as IL-13- secreting CD8 cells expressing GATA3 are increased in the skin of SSc patients. In vitro, TGFβ and Th2 cytokines promote collagen synthesis, whereas Th1 cytokines, such as IFNγ, rather counterbalance the fibrotic process (Figure 4C). Despite this findings, some reports support Th1 activation, in particular in the early stage of the disease[87, 88]. Our laboratory, together with other groups, has recently found that Th17 and Th22 cells are also upregulated in SSc. IL-17A and IL-22, hallmark cytokines of these subsets, appear to be involved in the inflammatory component rather than in the fibrotic manifestation of the disease, at least in humans [88, 89]. My contribution in the identification of Th17 and Th22 cells in SSc, as well in the definition of the role of IL-17 and IL-22 in fibrosis is discussed in sections 3.2 to 3.4 (references [18, 21, 51])

2.2.4. Psoriasis

Psoriasis is a frequent chronic inflammatory skin disease, affecting individuals of all age and gender. Several clinical forms exist, with plaque psoriasis (also known as psoriasis vulgaris) being the most common. This latter manifests with well-defined red plaques covered by silver scales, which characteristically appears at preferential sites, such as the elbows and knees, with a chronic recurrent course. Histological features of plaque psoriasis include acanthosis (thicker epidermis), hyperparakeratosis (retention of nuclei in a thicker cornified layer), and the presence of an important mononuclear infiltrate around dilated vessels. Acanthosis and hyperparakeratosis are observed as a result of the increased proliferation along with defects in terminal differentiation of keratinocytes. Neutrophils are also abundant and typically accumulate in the epidermis forming subcorneal microabscesses, also reffered to as Munro’s microabscesses [90, 91] (Figure 5A and B).

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18 Figure 5. Psoriasis. A) Typical skin lesion. B) Histological modifications of psoriatic lesional skin include achantosis (*), hyperparakeratose (**), perivascular immune cell infiltrate (***) and neutrophil collections in spinous/cornified layers (****). C) Scheme presenting a simplified view of the immunopathogenesis of psoriasis. (A – adapted from Boehncke and Shon, Psoriasis. Lancet 2015; B) Personal contribution; C) Adapted from Nestle et al., Psoriasis. N Engl J Med, 2009. 361(5): p.496-509)

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19 Although the skin is the most evident clinical manifestation, psoriasis has a systemic nature.

Psoriasis is associated with several comorbidities, such as psoriatic arthritis, metabolic syndrome, depression and cardiovascular events. Comorbidities seriously impact the overall quality of life of patients and reduce their life expectancy[92, 93]. In 2013 the Executive Board of WHO recommended to the 67th World Health Assembly a resolution to raise awareness of psoriasis as a major global health problem[94].

The immune involvement in psoriasis pathogenesis is well documented. Genome-wide associated scans reveal more than 40 loci associated with the disease. Most of them map to genes involved in immune function in general and in the IL-23/Th17 cell pathway in particular. The HLA-Cw6 locus accounts for almost 50% of the heritability, revealing the likely autoimmune nature of the disease[95]. Despite several efforts, the possible antigens driving the disease have not been fully identified. The antimicrobial peptide LL37, in complex with nucleic acids released by dying cells, has been proposed to cause the production of type I interferons by plasmacytoid and myeloid DCs, ultimately resulting in the activation of the IL-23/Th17 pathway[96, 97]. Th17 cells would travel back to the skin, where directly activate keratinocytes via the release of effector cytokines, such as IL-17A and IL-22.

Activated keratinocytes proliferate in an abnormal manner and release further inflammatory mediators and chemokines, such as IL-8 and TNF, which amplify the inflammatory response by creating an inflammatory loop (Figure 5C) [91]. More recently, the melanocyte antigen ADAMTS-like protein 5 (ADAMTSL5) has been proposed as an additional auto-antigen triggering IL-17A secretion from infiltrating CD8 T bearing HLA-Cw6 allele[98]. Several data obtained in vitro and in murine models of psoriasiform inflammation support the pathogenic role of Th17 cells and their related cytokines, especially IL-17A[29].

The clinical management of the psoriatic lesions relies since decades on the use of immunosuppressive agents, further underling the involvement of the immune system in its pathogenesis. Impressive amelioration of psoriasis skin manifestation has recently been achieved with the introduction of antibodies targeting the development of Th17 cells, or neutralizing the effector cytokine IL-17A[99-102]. This provides an additional insight of the key role of these pathways in the pathogenesis of the disease. Of interest, early responses to anti-IL-17A antibodies include the disappearance of neutrophils, simultaneously with skin normalization, while DCs and T cell numbers decreased only later[103]. This implies the existence of a pathogenically relevant and previously underestimated neutrophil-keratinocyte crosstalk. Besides, these results clearly establish psoriasis as a Th17- and more precisely IL- 17A-driven disease.

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20 Il-17A is part of a family comprising other 5 isoforms (IL-17B to IL-17F), as described in the section 2.1.2. IL-17 cytokines show several overlapping functions, whereas they may elicit different responses in a tissue-specific manner. Several of these cytokines have been linked to psoriasis pathogenesis, including IL-17F, IL-17C and IL-17E, although their precise role is still being assessed. [40, 104]. My contribution to the definition of the function of IL-17E in the pathogenesis of psoriasis will be further discussed is section 3.5 (reference [40]).

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21

3. SCIENTIFIC CONTRIBUTIONS

3.1. AhR drives the expansion of skin-homing Th22 cells in humans

Reference [47]: Brembilla NC, Ramirez JM, Chicheportiche R, Sorg O, Saurat JH, Chizzolini C. In vivo dioxin favors interleukin-22 production by human CD4+ T cells in an aryl hydrocarbon receptor (AhR)-dependent manner. PLoS One. 2011 Apr; 6(4): e18741

Personal contribution: I designed and performed all the experiments, analyzed the data and contributed to manuscript writing. I act as corresponding author.

The Aryl hydrocarbon receptor (AhR) is a ubiquitous cytoplasmic transcription factor activated by a wide range of planar aromatic hydrocarbons to which humans are frequently exposed. These include endogenous and exogenous ligands, with the latter being particularly prominent in industrialized countries. The pollutant 2,3,7,8-tetraclorodibenzo-p-dioxin (TCDD), or simply dioxin, is one of the most stable and high-affinity ligand of AhR. Dioxins are found in the environment as byproducts of waste incineration and steel industry, herbicide and pesticide usage, and exposure to cigarette smoke. Activation of AhR induces the transcription of several genes, among which xenobiotic response enzymes are the most studied. Between 2008 and 2010, our group, together with other laboratories, reported that AhR activation by TCDD or reversible agonists induced the production of IL-22 independently of IL-17A and IFNγ in human CD4 T cells in vitro[44, 48]. These cells have been later named Th22 cells.

In the following article, we assessed the immunological alterations caused by long-term exposure to TCDD in one individual who survived a deliberate poisoning with high dose of the pure compound (50’000 fold higher than the average exposition)[105]. We found that AhR activation in vivo favors the expansion of T cell that produce IL-22 in the absence of IL- 17A, IFNγ and IL-10, which shows a skin-homing phenotype. Of note, this patient suffered from a severe skin disease characterized by acne-like eruptions, better described as Metabolising Acquired Dioxin Induced Skin Hamartomas (MADISH).

This work corroborates previous results in vitro and was seminal to prove the existence of Th22 cells in humans. It provides further insight to the possible role of these cells in the controlling of skin homeostasis.

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In Vivo Dioxin Favors Interleukin-22 Production by Human CD4 + T Cells in an Aryl Hydrocarbon Receptor (AhR)-Dependent Manner

Nicolo` Costantino Brembilla¹*, Jean-Marie Ramirez¹, Rachel Chicheportiche¹, Olivier Sorg², Jean-Hilaire Saurat², Carlo Chizzolini¹

1Department of Immunology and Allergy, Swiss Centre for Applied Human Toxicology, University Hospital and School of Medicine, Geneva, Switzerland,2Department of Dermato-Toxicology, Swiss Centre for Applied Human Toxicology, University Hospital and School of Medicine, Geneva, Switzerland

Abstract

Background:The transcription factor aryl hydrocarbon receptor (AhR) mediates the effects of a group of chemicals known as dioxins, ubiquitously present in our environment. However, it is poorly known how the in vivoexposure to these chemicals affects in humans the adaptive immune response. We therefore assessed the functional phenotype of T cells from an individual who developed a severe cutaneous and systemic syndrome after having been exposed to an extremely high dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Methodology/Principal Findings: T cells of the TCDD-exposed individual were studied for their capacity to produce cytokines in response to polyclonal and superantigenic stimulation, and for the expression of chemokine receptors involved in skin homing. The supernatants from T cells of the exposed individual contained a substantially increased amount of interleukin (IL)-22 but not of IL-17A, interferon (IFN)-cor IL-10 when compared to nine healthy controls.In vitroexperiments confirmed a direct, AhR-dependent, enhancing effect of TCDD on IL-22 production by CD4+ T cells. The increased production of IL-22 was not dependent on AhR occupancy by residual TCDD molecules, as demonstrated in competition experiments with the specific AhR antagonist CH-223191. In contrast, it was due to an increased frequency of IL-22 single producing cells accompanied by an increased percentage of cells expressing the skin-homing chemokine receptors CCR6 and CCR4, identified through a multiparameter flow cytometry approach. Of interest, the frequency of CD4+CD25^hiFoxP3+T regulatory cells was similar in the TCDD-exposed and healthy individuals.

Conclusions/Significance:This case strongly supports the contention that human exposure to persistent AhR ligandsin vivoinduce a long-lasting effect on the human adaptive immune system and specifically polarizes CD4+T cells to produce IL-22 and not other T cell cytokines with no effect on T regulatory cells.

Citation:Brembilla NC, Ramirez J-M, Chicheportiche R, Sorg O, Saurat J-H, et al. (2011)In VivoDioxin Favors Interleukin-22 Production by Human CD4+T Cells in an Aryl Hydrocarbon Receptor (AhR)-Dependent Manner. PLoS ONE 6(4): e18741. doi:10.1371/journal.pone.0018741

Editor:Bernhard Ryffel, French National Centre for Scientific Research, France ReceivedNovember 9, 2010;AcceptedMarch 16, 2011;PublishedApril 15, 2011

Copyright:!2011 Brembilla et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding:This work was supported by grant 31003A_124941/1 from the Swiss National Science Foundation to C.C. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.

Competing Interests:The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent member of a group of halogenated aromatic hydrocarbons, generally known as dioxins [1]. Dioxins are produced when organic material is burned in the presence of chlorine and are therefore widely implicated in many industrial as well as natural processes. Major sources of environmental dioxins include waste incinerators and steel industry as well as the use of herbicide and pesticide containing chlorophenols. Due to their high lipophilicity and poor metabolism, dioxins accumulate in lipid-rich tissues of animals and rapidly climb the food chain up to humans [2]. In addition, dioxins are presents in cigarette smoke. As a conse- quence, concentrations of dioxins are found in all humans, with higher levels commonly identified in persons living in industrialized countries.

TCDD, considered as the prototypical dioxin, has been shown to have pleiotropic biological effects at low doses in multiple animal species [3,4]. The majority of TCDD effects are mediated via binding and activation of the intracellular aryl hydrocarbon receptor (AhR), as demonstrated by the loss of responsiveness to TCDD in AhR knockout mice [5]. The elevated toxicity of TCDD is caused by its extremely high affinity for AhR and its long half- life (5–10 years in humans [6]). Upon ligand binding AhR undergoes a conformational change and translocates into the nucleus, where it dimerizes with the AhR nuclear translocator (ARNT) and regulates, by binding to xenobiotic response elements (XRE), the expression of a variety of genes, including the xenobiotic metabolizing enzymeCYP1A1(cytochrome P450) [7].

In mice, AhR activation is reported to regulate T helper (Th) 17 and T regulatory (Treg) cell differentiation and to modulate immune responses to experimental induced encephalomyelitis in a

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ligand-dependent manner [8,9,10]. In addition, AhR has been shown to be crucial for interleukin (IL)-22 expression [10] and a regulatory mechanism for IL-22 production via a Notch-AhR axis has been identified [11]. We [12] and others [13] have demonstrated a role for AhR agonists, including TCDD, in promoting thein vitro production of IL-22 but not of IL-17 by human CD4 T cells. The possibility that in humans AhR stimulation could participate to thein vitrodifferentiation of IL- 10-producing Treg cells has also been suggested [14].

IL-22 is a member of the IL-10 family of cytokines and signals via a receptor consisting of IL-22R and IL-10R2 subunits. IL-22 does not serve the communication between immune cells since cells of hematopoietic origin do not express IL-22R [15]. It mainly acts on epithelial cells of the gastrointestinal tract and the skin, where it promotes antimicrobial defense, protection against damage and regeneration [16]. However, its role in chronic inflammatory disorders may be either protective [17] or highly pathogenetic [18,19]. T cells able to produce IL-22 in the absence of IL-17 and interferon (IFN)-c, have been named Th22 cells and are enriched in cells expressing the skin-homing chemokine receptors CCR6, CCR4 and CCR10 while lacking CXCR3 [13,20]. Th22 cells have been identified in the skin of individuals suffering of psoriasis and atopic dermatitis [21,22,23] and are thought to be important in skin immunosurveillance and immunopathology [13,20,21].

All the data on the effects of AhR ligands on human T cell subsets have been so far generatedin vitroand their relevance toin vivo situations remains largely unknown. In this report, we extensively characterize the long-term immunological modifications induced by TCDD in one of the two ever reported cases of a human being who survived thein vivo exposure to an extremely high dose of the pure compound [24]. Our data indicate for the first time that in vivo exposure to TCDD induces a selective increase in the frequency of T cells producing IL-22 but neither IL-17, IL-10, nor IFN-c, which preferentially express skin-homing chemokine receptors. These data strongly support the in vivo existence in humans of Th22 cells that depend on AhR for their expansion.

Materials and Methods Patient

We obtained written approval from the patient to release peer- reviewed scientific information about his case. Our patient had been intoxicated by pure dioxin (TCDD) presumably in late 2004 at the age of 50. In early January 2005 he arrived under controlled conditions at the Geneva University Hospital, Switzerland, where we identified a TCDD concentration of 108,000 pg/g of lipid weight in his blood serum [24]. Similar levels were identified by an independent laboratory in a sample taken from the same patient in mid-December 2004 [25]. This concentration was more than 50,000 times the averaged TCDD in the general population (normal value: 10–20 pg/g of lipid weight) [26]. The patient was suffering from a severe skin disease, the historically called

‘‘chloracne’’, consisting in what we call now ‘‘metabolizing acquired dioxin-induced skin hamartomas’’ (MADISH) to describe his skin condition [27]. All the experiments shown in this report were performed 4 years after the acute exposure to TCDD, when the patient had a TCDD concentration of 19,000 pg/g of lipid weight in his blood serum and no TCDD-related pathology was anymore clinically apparent. Nine sex and age (52610 years) matched healthy members of the laboratory served as controls.

CD4 and CD8 T cell frequencies in the peripheral blood of the TCDD-exposed individual were within the range of healthy

controls (CD4+CD3+ T cells: 62.4% and 6167% of living lymphocytes, respectively; CD3+CD8+ T cells: 25.7% and 1867% of living lymphocytes, respectively). Permission to perform this investigation was granted by Comite´ departemental de me´decine interne et me´decine communaute`re des hoˆpitaux universitaires de Gene`ve. Written informed consent according to the Helsinki declaration was obtained from each individual involved in this study.

Reagents

Fetal calf serum (FCS), phorbol myristate acetate (PMA), b- mercaptoethanol, staphylococcal enterotoxin B (SEB) and brefeldin A were from Sigma Chemicals (St. Louis, MO); TCDD from Cambridge Isotope Laboratories (Andover, MA); ionomycin from Calbiochem (Merck KGaA, Darmstadt, Germany); RPMI 1640 medium, phosphate buffered saline (PBS), penicillin, streptomycin, L-glutamin, nonessential amino acids, sodium pyruvate from Life Technologies (Carlsbad, CA); human rIL-2 from Biogen (Zug, Switzerland); anti-IL-22-PE from R&D (Minneapolis, MN); anti- CD28 (CD28.2), anti-CD45RA-FITC, anti-CCR6-PercPCy5.5, anti-CCR4-PECy7, anti-CXCR3-APC, anti-CD4-PE-Cy5, anti- CD4-FITC, anti-CD4-APC-Cy7, anti-CD8-APC-Cy7 and anti- CD3-FITC from BD (Franklin Lakes, NJ); anti-IL-17A-FITC, anti-IL10-Alexa 488 and anti-IFN-c-PE-Cy7 from Biolegend (San Diego, CA); anti-CD25-APC and anti-FoxP3-PE from eBioscience (San Diego, CA); anti-CD3 (OKT3) Ab from ATCC (Manassas, VA); CH-223191 from VWR (Dietikon, Switzerland).

Cell culture

Peripheral blood mononuclear cells (PBMC) were purified by Ficoll-paque Plus (GE Healthcare, Pittsburgh, PA) gradient centrifugation and frozen in liquid nitrogen until use or processed immediately (experiment in Fig. 1A). The cells from the TCDD- exposed individual were treated under the same experimental conditions and in parallel to that of healthy individuals in all experiments shown in this manuscript. PBMC were rested o/n in RPMI 1640 medium supplemented with 10% FCS (complete RPMI, cRPMI) as described [28] and then used in short term (up to 24 h) or long term (7 d) cultures. In short term cultures PBMC were activated for 4.5 h by PMA (50 ng/ml) and ionomycin (1mM) or for 24 hours in flat-bottom 96-well plates in presence of coated anti-CD3 mAb (1mg/ml) and soluble anti-CD28 mAb (1mg/ml). When intracellular cytokine determination was performed, brefeldin A (2.5mg/ml) was added after 1.5 and 3 hours from the beginning of the activation, respectively. In long-term activation experiments, PBMC were cultured at 1610⁶cells/ml in 24-well plates in cRPMI in the presence of soluble anti-CD3 Ab (0.1mg/ml) or SEB (0.2mg/ml). Culture medium was supplemented with IL-23 (10 ng/ml) and IL-2 (20 U/ml) was added 48 h after culture initiation. When used, TCDD (10 nM, unless otherwise specified) and CH-223191 (3mM) were added at the beginning of the culture. At day 7 of culture, supernatants were collected and frozen until cytokine determination and the cells were harvested and activated for 4.5 h by PMA and ionomycin for FACS analysis.

Flow cytometry and cytokine assays

Cell surface and intracellular staining were assessed by FACS analysis using FACSCanto (BD) and data were analyzed by FlowJo software 7.5 (Tree Star). For intracellular cytokine determination, cells were stained with anti-CD4-PE-Cy5 or anti- CD4-APC-Cy7 or anti-CD3-FITC mAbs, fixed and stained with anti-IL-17A-FITC or anti-IL-10-Alexa 488, anti-IL-22-PE, anti- IFN-c-PE-Cy7 and anti-IL-4-APC mAbs using BD Cytofix/

In VivoDioxin Favors Th22 Cells Expansion

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The different dimensions of how inflammation impacts cutaneous biology

Thesis

Reference

The different dimensions of how inflammation impacts cutaneous biology

The different dimensions of how inflammation impacts cutaneous biology

ABBREVIATIONS used in the text

TABLE OF CONTENTS

1. SUMMARY

2. INTRODUCTION

3. SCIENTIFIC CONTRIBUTIONS

In Vivo Dioxin Favors Interleukin-22 Production by Human CD4 + T Cells in an Aryl Hydrocarbon Receptor (AhR)-Dependent Manner