Dissertation
Zur Erlangung des akademischen Grades Doktor rerum naturalium
(Dr. rer. nat) im Fach Biologie eingereicht an der
Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin
von
M.Sc. Vandana Kumari
Präsident der Humboldt-Universität zu Berlin Prof. Dr. Jan-Hendrik Olbertz
Dekan der Lebenswissenschaftlichen Fakultät Prof. Dr. Richard Lucius
Gutacher/innen: 1. Prof. Dr. A. Radbruch 2. Prof. Dr. M. Worm 3. Prof. Dr. P. Franken
Tag der mündlichen Prüfung: 21.04.2015
ALL THAT WE ARE IS THE RESULT OF ALL THAT WE HAVE THOUGHT.
- BUDDHA
TABLE OF CONTENTS
LIST OF ABBREVIATIONS ... 6
ABSTRACT ... 10
ZUSAMMENFASSUNG ... 11
1. INTRODUCTION ... 12
1.1. ANATOMICAL SKIN STRUCTURE ... 12
1.2. SKIN BARRIER AND IT’S DISRUPTION IN SKIN PATHOLOGY ... 14
1.2.1 Physical and chemical irritants ... 15
1.2.2 Contact dermatitis (CD) and Atopic dermatitis (AD) ... 16
1.3. KERATINOCYTES ... 21
1.3.1 Role of keratinocytes in skin irritation ... 22
1.3.2 Role of keratinocytes in AD ... 23
1.4 TUMOR NECROSIS FACTOR-α (TNF-α) ... 24
1.4.1 TNF-α – a proinflammatory cytokine... 24
1.4.2 Role of TNF-α in skin irritation ... 25
1.4.3 Role of TNF-α in AD ... 26
1.5 THYMIC STROMAL LYMPHOPOIETIN (TSLP) ... 27
1.5.1 Role of TSLP in skin irritation ... 28
1.5.2 Role of TSLP in AD ... 30
1.6 OBJECTIVES ... 31
2. MATERIAL AND METHODS ... 32
2.1 MATERIALS ... 32
2.2 METHODS ... 36
2.2.1 Animal experiments ... 36
2.2.2 Cell culture methods ... 41
2.2.3 TSLP enzyme linked immunosorbent assay (ELISA) ... 43
2.2.4 RNA isolation ... 44
2.2.5 Reverse transcription ... 44
2.2.6 Real-time polymerase chain reaction ... 45 3
2.2.7 Isolation and culture of bone marrow cells and generation of bone marrow-
derived mast cells (BMcMCs) ... 47
2.2.8 Flow cytometry ... 48
2.2.9 Stimulation of BMcMCs ... 49
2.2.10 Histology and immunohistochemistry ... 49
2.3 STATISTICAL ANALYSIS ... 52
3. RESULTS ... 53
3.1 SKIN IRRITATION LEADS TO TSLP PRODUCTION ... 53
3.1.1 Physical or chemical irritation of the skin leads to production of TSLP in vivo . 53 3.1.2 Pro-inflammatory cytokines elevate TSLP production in murine KCs ... 55
3.1.3 Skin biopsies from mouse and human produce TSLP ex vivo ... 57
3.1.4 IL-1 contributes to SDS-mediated TSLP induction ... 58
3.2 AGGRAVATED AD IN TNF-/- MICE ... 59
3.3 ROLE OF TSLP IN AD AGGRAVATION UPON TNF DEFICIENCY ... 60
3.3.1 Increased TSLP levels in lesional skin of TNF-/- mice and correlation with AD severity ... 60
3.3.2 Anti-TSLP protect TNF-/- regarding AD onset ... 62
3.4. ENDOGENOUS TNF-α DOES NOT CONTRIBUTE TO TSLP PRODUCTION ... 63
3.5 MAST CELLS CONTRIBUTE TO TSLP PRODUCTION ... 65
3.5.1 MCs are increased in lesional skin of TNF-/- mice and correlate with AD and TSLP ... 65
3.5.2 Anti c-Kit is protective for AD development in TNF-/- mice ... 66
3.5.3 MCs do not produce a relevant amount of TSLP ... 67
3.5.4 MCs as instructors of TSLP production by KCs ... 68
4. DISCUSSION ... 71
4.1 SKIN IRRITATION LEADS TO RAPID INDUCTION OF TSLP, INDEPENDENT FROM TNF-α, BUT PARTIALLY DEPENDS ON IL-1 ... 71
4.2 TNF-/- MICE DEVELOP AGGRAVATED AD AND DISPLAY INCREASED TSLP EXPRESSION AND MCs NUMBERS CORRELATING WITH DISEASE SEVERITY ... 76
4.3 ENHANCED TSLP LEADS TO AD MANIFESTATION ... 79
4.4 MCs SEEM TO PLAY A ROLE BETWEEN TNF-DEFICIENCY AND TSLP ... 81 4
4.5 CONCLUSION AND OUTLOOK ... 84
REFERENCES ... 87
APPENDIX ... 99
ACKNOWLEDGEMENTS ... 101
SELBSTÄNDIGKEITSERKLÄRUNG / DECLARATION ... 103
5
LIST OF ABBREVIATIONS -/-
αh αm ANOVA
AD e.c
β-Me BMcMCs
bp BSA C57BL/6
CASY CCL CD
DNA cDNA
dsDNA
CLA CT
CXCL8 DC
dDCs EDTA
ELISA ERK
FACS
FBS Fc
FcεRI Fig FITC
Knockout Anti-human Anti-mouse
Analysis of variance Atopic dermatitis Epicutaneous β-mercaptoethanol
Bone marrow cultured mast cells Base pair
Bovine serum albumin C57 black 6
CASY® Cell Counter Chemokine ligand Cluster of differentiation Desoxyribonucleic acid Copy desoxyribonucleic acid Double-Stranded DNA
Cutaneous lymphocyte-associated antigen Threshold cycle value
CXC ligand 8 Dendritic cell
Dermal dendritic cells
Ethylenediaminetetraacetic acid Enzyme linked immunosorbent assay Extracellular signal-regulated kinase Fluorescence activated cell sorter Fetal Bovine Serum
Fragment crystallizable of Ig Fc epsilon receptor I
Figure
Fluorescein IsoThioCyanate
6
g
GM-CSF H1R
H2O2
H4R HCl
HMGB1 HPRT
hrs HRP IFNγ Ig
ICAM-1 IL-
IL-7Rα IL-1Ra IMDM i.p i.d LSAB2
JAK JNK KCs
kDa LTα LTC4
MΦ MACS MAP MCs
MDM2 MgCl2
Acceleration of gravity
Granulocyte-macrophage colony-stimulating factor Histamine 1 receptor
Hydrogen peroxide Histamine 4 receptor Hydrochloric acid
High mobility group box chromosomal Protein 1 Hypoxanthine-guanine phosphoribosyltransferase Hours
Horseradish peroxidase Interferon gamma
Immunoglobulin
Intercellular adhesion molecule-1 Interleukin-
Interleukin-7 receptor alpha Interleukin-1 receptor antagonist Iscove's Modified Dulbecco's Medium Intraperitoneal
Intradermal
Labelled Streptavidin-Biotin2 System- Janus Activated Kinase
c-Jun N-terminal kinases Keratinocytes
Kilodalton Lymphotoxin α Leukotriene C4 Macrophage
Magnetic Cell Sorting Mitogen-activated protein Mast cells
Murine double minute 2 Magnesium Chloride
7
mMCP6 mRNA NF-κB
NHBE NK
O.C.T OVA p38 PBS PBST PCR
PE Pen/Strep
PGD2 Plcb 3
PMA Poly I:C RANTES rh rm
RNA rpm RT
SB
SEM SC SCF
SCORAD SDS
SG SLS SS
Mouse Mast Cell Protease 6 Messenger ribonucleic acid
Nuclear factor kappa-light-chain-enhancer of activated B cells Normal Human Bronchial Epithelial
Natural killer
Optimal Cutting Temperature Ovalbumin
Phospho 38
Phosphate buffered saline
Phosphate buffered saline + Tween-20 Polymerase chain reaction
phycoerythrin
Penicillin and streptomycin Prostaglandin D2
Phospholipase C-Beta 3 Phorbol Myristate Acetate Polyinosinic:polycytidylic acid
Regulated on Activation Normal T Cell Expressed and Secreted Recombinant human
Recombinant mouse Ribonucleic acid
Revolutions per minute Reverse transcriptase Stratum basale
Standard error of the mean Stratum corneum
Stem cell factor
Severity Scoring of Atopic Dermatitis Sodium dodecyl sulphate
Stratum granulosum Sodium lauryl sulphate Stratum spinosum
8
STAT6 TAE
TBS TEWL TGF-β Th
TLR TNF-α
TNFR
TPA Treg TSLP TSLPR
qPCR UTR UV wt
Signal Transducers and Activators of Transcription 6 TRIS-Acetat-EDTA
Tris-buffered saline
Transepidermal water loss Transforming growth factor beta T-helper
Toll like receptor
Tumor necrosis factor-α
Tumor necrosis factor receptor
12-o-Tetradecanoylphorbol-13- acetate Regulatory T cell
Thymic stromal lymphopoietin
Thymic stromal lymphopoietin receptor quantitative PCR
Untranslated region Ultraviolet
Wildtype (C57BL/6)
9
ABSTRACT
The skin is the largest organ of an individuum and builds the barrier for a host against the environment. Skin barrier disruption by exogenous or endogenous stimuli can lead to skin inflammation. As a consequence, irritant or atopic eczema, frequent skin diseases, may evolve. Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine which plays a central role in inflammatory processes.
The main aim of this thesis was to clarify whether and how endogenous TNF-α is contributing to skin inflammation driven by exogenous and endogenous triggers.
The role of endogenous TNF-α was studied using TNF knockout (-/-) mice. In an irritation model, chemical and physical stimuli were applied on to mouse skin.
Thymic stromal lymphopoietin (TSLP) was significantly induced by the used irritants. This TSLP induction was independent from endogenous TNF-α proven by using TNF-/- mice.
Next the role of TNF-α in atopic dermatitis (AD) promoting an allergic skin inflammation was investigated. TNF-/- mice developed more severe AD compared to the wildtype mice and TSLP was significantly increased and correlated with the severity of the eczema. To prove the pathophysiological role of TSLP for AD progression, TNF-/- mice were pretreated with an TSLP antibody. Indeed, these mice developed less AD symptoms compared to the control mice.
Mast cells (MCs) were also significantly increased in lesional skin in the AD model and moreover, correlated with AD severity, but also with TSLP expression.
10
ZUSAMMENFASSUNG
Die Haut ist das größte Organ des Menschen und bildet die Barriere gegenüber Einwirkungen aus der Umwelt. Die Störung der Hautbarriere durch exogene und endogene Reize führt zu einer Entzündungsreaktion in der Haut. In der Folge können Hauterkrankungen wie die irritative oder Atopische Dermatitis entstehen.
Der Tumor Nekrose Faktor-α (TNF-α) ist ein pleiotrop wirksames Zytokin, das eine zentrale Rolle bei entzündlichen Prozessen spielt.
Ziel der vorgelegten Promotionsarbeit war zu untersuchen, ob und wie TNF-α zu Entzündungsgeschehen, ausgelöst durch exogene und endogene Faktoren, beiträgt.
Die Bedeutung von TNF-α wurde in TNF-ko Mäusen in verschiedenen Hautmodellen untersucht. Für das Irritationsmodell wurden chemische und physikalische Reize verwendet. TSLP (Thymic stromal lymphopoietin) wurde durch die verschiedenen Stimuli signifikant induziert. Diese Induktion war unabhängig von der endogenen TNF-α Produktion, gezeigt durch den Einsatz von TNF- ko Mäusen . Da endogenes TNF-α für die Hautirritation keine notwendige Bedingung darstellte, wurde die Bedeutung von TNF-α bei der atopischen Dermatitis (AD) untersucht.
TNF-α defiziente Mäuse zeigen verstärkt Ekzeme im Vergleich zu Wildtyp Mäusen.
Die Behandlung von TNF-ko Mäusen mit einem TSLP Antikörper führte zu einer Verminderung des Ekzems.
Mastzellen wurden vermehrt in läsionaler Haut gefunden und korrelierten mit dem Schweregrad des atopischen Ekzems sowie der TSLP-Expression.
Schlagwörter:
Tumor Nekrose Faktor-α, Thymic stromal lymphopoietin, Hauterkrankungen, Atopischen Dermatitis, Mastzellen
Keywords:
Tumor necrosis factor-α, Thymic stromal lymphopoietin, skin inflammation, Atopic dermatitis, Mast cell
11
1. INTRODUCTION
1.1. ANATOMICAL SKIN STRUCTURE
The skin provides a protective barrier between the inner and outer environment to protect an individuum from various potential dangerous microbes1. The skin is composed of three layers, the epidermis, dermis and subcutis2. The epidermis is of highest importance for the skin barrier integrity and provides an individuum with physical, chemical or biochemical barriers. The epidermis is formed by several layers of keratinocytes which undergo a differentiation process. These are the stratum basale, the stratum spinosum, the stratum granulosum and the stratum corneum (Fig. 1)1,3,4. The stratum basale is the layer which contains basal stem cells that are capable to proliferate into keratinocytes and can amplify the cell numbers5. The stratum spinosum is characterized by visible desmosomes, which contribute to the appearance of spindle shaped cells. These cells express the early differentiation marker cytokeratin 10. The differentiation of cells can be seen from bottom to top, by the presence of intermediate differentiation marker involucrin in the upper spinous cell layers but not in the lower ones5. The skin core is mainly composed of a continuous sheet of flat anucleated corneocytes which represent differentiated keratinocytes of the outer layer of stratum granulosum containing keratin filaments1,3,4. The stratum granulosum consist of 3–5 cell layers and is characterized by lamellar bodies and keratohyalin granules. These layered of cells express and process the two late differentiation markers filaggrin and loricrin6. The primary skin barrier is mainly provided by stratum corneum layer as a robust barrier against the percutaneous penetration of chemicals and microbes, but also mechanical injuries1,7. Cells in the stratum corneum layers are connected together by lipid bilayers, which forms a brick-like structure which form an insoluble, rigid structure referred to as cornified envelope. The stratum corneum is also responsible in different active processes such as regulation of water loss from the skin to the outer atmosphere, known as transepidermal water loss (TEWL)1,7.
12
Figure 1: Anatomical skin structure including the epidermal layers.
The skin structure is complex and enables to build a barrier against environment. The epidermis contains stratum corneum followed by stratum lucidum, stratum granulosum, stratum spinosum and stratum basale. The dermis is mainly composed of collagen, elastic tissue and reticular fibres. It contains many different cell types such as dendritic cells (DCs), T cells subsets, fibroblast, macrophages and mast cells (MC) (not shown). The subcutis is composed of the adipose tissue.
Adopted from Skin barrier function and its importance at the start of the atopic march, Mary Beth Hogan, Kathy Peele, and Nevin W. Wilson, Journal of Allergy (2012).
The dermis forms the thickest structure of the skin containing sebaceous glands, sweat glands and hair follicles8,9. The dermis is formed by connective tissue and a network of capillaries and blood vessels. Dilatation or constriction of these blood vessels and capillaries provides thermoregulation to the body10. The dermis also provides elasticity to the skin as it contains elastin fibers and collagen11. By contrast, the epidermis contains tight junctions, adherens junctions, desmosomes, gap junctions and keratins filaments to form the skin barrier12. Tight junctions are the cell to cell junctions which regulate paracellular activities of molecules and are responsible for the separation of the apical from the basolateral part of the cell membrane, reducing the diffusion of proteins and lipids between the cells. Tight junctions and desmosomes play a vital role in the stabilization of the cell to cell adhesion, to maintain the cell shape and the tissue integrity. Gap junctions are 13
important for cell to cell interaction. The major components of gap junctions are connexins, which homo- or heteromerize to connexons to form channels, which allow the passage of ions and small molecules between cells1. Keratins are the most abundant structural proteins synthesized by keratinocytes that assemble throughout the cytoplasm and terminate at desmosomes1,9.
1.2. SKIN BARRIER AND IT’S DISRUPTION IN SKIN PATHOLOGY
The skin is a metabolically active organ. Different physiological processes support to maintain the skin barrier10. The primary function of the skin is to protect inner body from physical, chemical, thermal or mechanical hazards as well as the invasion of microorganisms (Fig. 2)1. It also reduces the harmful effects of UV radiation and acts as a sensory organ (Fig. 2)10. To maintain the function of the skin barrier, a large number of factors are required. These include an cell to cell interaction within epidermis, the prevention of excessive water loss, the communication with the immune system and the renewal of the skin cells. When the epidermal skin barrier is disrupted, the initial response to cellular damage of the epidermal cells is a stimulation signal to replace the damaged cells13 and to maintain the skin homeostasis. The skin-resident immune cells such as epidermal langerhans cells or dendritic cells are key players in restoring the homeostasis14. Upon skin injury, KCs start producing pro-inflamamatory cytokines such as Interleukin-1β (IL-1β), IL-6, IL-18 and TNF-α, which further activate dermal dendritic cells (DCs) in the presence or absence of antigen. Upon stress signalling, KCs gets activated and contribute to dermal DC activation by releasing interferon-α (IFN-α) (Fig. 2). Activated dermal DCs promote the proliferation of skin-resident T cells i.e.
CD4+ or CD8+ T cells (Fig. 2). Stimulated T cell further produce pro-inflammatory cytokines and chemokines which stimulate epithelial and mesenchymal cells e.g.
keratinocytes and fibroblasts thus amplifying the inflammatory reaction in the skin (Fig. 2)14.
14
Figure 2: Disrupted skin barrier leads to inflammatory response skin.
Exposure to irritants, UV light or infections agent’s leads to barrier disruption is triggering the immune response to retain the skin homeostasis. Upon stimulation keratinocytes produce proinflammatory cytokines such as TNF- α, IL-1β, TSLP, which further promote the transition of dermal dendritic cells (dDCs) and activate MCs and T- cells.
Adapted from Skin immune sentinels in health and disease. Frank O. Nestle, Paola Di Meglio, Jian-Zhong Qin and Brian J. Nickoloff, Nat Rev Immunol. Oct 2009; 9(10): 679–691.
1.2.1 Physical and chemical irritants
Exposure of the skin to different irritants can lead to an impairment of the barrier function and a consecutive damage of the epidermal cells15. Many studies have been done to understand the mechanism of acute and chronic irritation16. As it is difficult for ethical reasons to study the pathogenesis of irritation at a cellular level in humans, mouse models have been used to study the physico-chemical events during these reactions. Many studies have been performed using different irritants such as sodium dodecyl sulphate (SDS), acetone, croton oil or tape stripping17. Measurements to assess a disturbed skin barrier include TEWL, electrical
15
capacitance (stratum corneum hydration), percutaneous drug transport, and skin color reflectance (erythema)17,18. Willis CM et al. observed that irritation with 5%
SDS for 48 hrs resulted a strong inflammatory response with the onset of increased numbers of infiltrating cells consisting polymorphonuclear leukocytes and mononuclear cells19. Another group has shown that higher concentrations of SDS resulted in a down regulation of HLA-DR expression on Langerhans cells20. Another common irritation method which is widely used for the induction of barrier disruption with less cytopathic effects on keratinocytes is tape stripping. With the aid of adhesive tape strips, the layers of the stratum corneum were removed after 30times tape stripping21. Disruption of stratum corneum leads to an increase of the TEWL and induces the production of different inflammatory mediators17,22. Such induction of a proinflammatory immune response in human keratinocytes has been shown by different irritants such as croton oil, phenol and SLS as published by Wilmer et al.
(1994)23. In particular croton oil and phenol directly induce the expression of IL-18 without the intermediate production of IL-1α and TNF-α23.
1.2.2 Contact dermatitis (CD) and Atopic dermatitis (AD) Contact dermatitis
Contact dermatitis is an inflammatory response of the skin characterized by erythematous and pruritic skin lesions that occur after direct contact with exogenous substances24. Contact dermatitis is frequent and a main cause of occupational dermatitis25. Based on the pathophysiology contact dermatitis is classified in two subtypes: irritant contact dermatitis (ICD) and allergic contact dermatitis (ACD)24. Even though it is possible to differentiate between ICD from ACD at clinical levels, both manifestations can have similar clinical and histological presentations26.
Irritant contact dermatitis (ICD)
Irritant contact dermatitis is considered as the most common type of contact dermatitis26. It is the consequence of an activated innate immune response of skin to various physical and chemical stimuli. It occurs in response of skin injury by foreign particle without prior immunological sensitization of the skin. The
16
development of ICD depends on a complex interplay between endo- and exogenous factors27. Intrinsic factors which influence development of ICD include genetic predisposition eg. age, sex and body area, whereas extrinsic factor include the type of the irritant, the irritant concentration and the time of exposure27. An impairment of the skin horny layer and epidermal cell damage are considered to be the main factors in the pathogenesis of ICD. The underlying mechanism of ICD includes an activation of the innate immune response with the release of IL-1α, IL- 1β, TNF-α, GM-CSF and IL-8 (Fig. 3A)28. Consecutively, these cytokines activate Langerhans cells (LC), dDCs and endothelial cells, which further support the cellular recruitment at the site of damage e.g. lymphocytes, macrophages, neutrophilis (Fig.
3A). These cellular infiltrates further promote the inflammatory pathway (Fig. 3A)28. Allergic contact dermatitis (ACD)
Allergic contact dermatitis is a delayed hypersensitivity reaction mediated by antigen-specific T cells29. It occurs only in sensitized patients i.e. individuals who have build an immunological memory response upon a prior contact. The concentration of an allergen is important to initiate an ACD26. ACD is characterized by pruritic papules and vesicles on an erythematous base, in the chronic condition lichenified pruritic plaques can be present. Individuals with a history of ACD develop the symptoms a few days after exposure in the area that was in direct contact with the allergen30. Similar to the scenario in ICD the allergen exposure result in an activation of the innate immune system through a release of proinflammatory cytokines by KC including IL-1α, IL-1β, TNF-α, GM-CSF, IL-8 and IL-18 with in consequence the onset of vasodilation and cellular recruitment (Fig. 3B)28. Upon contact with allergens, LCs and dDCs migrate to the draining lymph nodes, where they activate allergen-specific T cells e.g. Th1, Th2, Th17 and regulatory T (Treg) cells (Fig. 3B)28. Activated T cells further proliferate and enter into the circulation and reach to the site of initial exposure, along with other immune cell such as mast cells and eosinophils (Fig. 3B). Once an individual is re-exposed to an allergen, the allergen-specific T cells, along with other inflammatory cells, enter the site of exposure and release proinflammatory cytokines which consequently stimulate the KCs to induce an inflammatory cascade (Fig. 3B)28.
17
Figure 3. Pathogenesis of irritant contact dermatitis (ICD) and allergic contact dermatitis (ACD).
A) In ICD, encounter with an irritant stimulate KCs by activating innate immunity with the release of pronflammatory cytokines such as IL-1α, IL-1β, TNF-α etc. from epidermal KCs. These cytokines further activate inflammatory cells e.g. LCs, dDCs, and endothelial cells, all of which contribute to cellular recruitment to the site of KC damage and further initiate the inflammatory cascade.
B) During sensitization phase of ACD, allergens activate innate immunity through KC activation and proinflammatory cytokines release as well as with vasodilation, cellular recruitment, and infiltration. Upon exposure to allergen, LCs and dDCs migrate to the lymph nodes, where they activate allergen-specific T cells e.g. Th1, Th2, Th17, and regulatory T (Treg) cells. Activated T cells proliferate and reach to the site of infection along with other cell types such as mast cells and eosinophils. Upon re-encountering with allergen, the hapten- specific T cells get activated and along with other inflammatory cells, enter the site of exposure and release proinflammatory cytokines and subsequently stimulate KCs to induce an inflammatory cascade.
Reprinted from Dhingra et al. 2013: Mechanisms of contact sensitization offer insights into the role of barrier defects vs. intrinsic immune abnormalities as drivers of atopic dermatitis, J Invest Dermatol.2311-4. (Oct 1, 2013.). Copyright (2014), with permission from Nature publishing group.
18
Atopic dermatitis
AD is a chronic-relapsing, eczematous skin disease clinically characterized by erythema, edema, excoriation, xerosis, intense pruritus and a typical localization pattern31. Commonly, AD initiates early in childhood (i.e. early-onset AD)31,32. Epidemiological studies point towards an increase in AD prevalence in the last decades affecting around 10-20% of children and 1-3% of the adult population worldwide32-34.
Pathophysiology of atopic dermatitis
AD is a highly complex inflammatory skin disease which depends on the interplay between genetic and environmental factors35. The understanding of AD development is still not completely clear especially at the molecular level36. It is still not certain whether AD is a consequence of an immune dysfunctioning or due to genetic defects or both31,32,36,37. A defect of the skin barrier function plays a crucial role in the pathogenesis of the disease. It leads to an increase of the epidermal water loss and a promotion of an invasion of allergens, microbes or any other irritants (Fig. 4)38. Different studies have shown that a defect of skin barrier promotes skin inflammation in AD patients34,39. Filaggrin an important skin barrier protein was identified to play a significant role in AD progression. Around 20% of AD patients display a null mutation in the gene encoding for filaggrin34,35,40. The presence of the filaggrin gene mutation has shown to increase skin dryness in AD patients41. Different cytokines such as IL-4, IL-13 and TNF-α have been shown to reduce the expression level of filaggrin in AD patients as well42. Among filaggrin several other proteins are involved in forming the skin barrier and may be relevant in AD as well. Moreover patients even though carrying filaggrin mutations can outgrow the disease suggesting that breakdown in the skin barrier is not sufficient for the development of AD43,44.
Various studies have shown that different immune cells are involved in the AD progression apart from the skin barrier. T cell plays a major role in the AD development especially at the early stage of the disease where an increased Th2 response is responsible for the major immune dysbalance45. Data from both human 19
and mouse studies show that CD4+ T cells are involved in the development of AD31,37,46. Specific DCs in the skin including epidermal Langerhans cells and inflammatory dendritic cells activate T cells38. In acute and chronic AD lesions, the expression levels of T cell induced cytokines i.e. IL-4, IL-5 and IL-13 were significantly increased (Fig. 4). Several studies indicate that also the other T-cell types such as T-reg, Th17, Th 9 and Th 22 are involved in the pathogenesis of AD but their exact role in the AD progression is still not clear (Fig. 4)47,48. Keratinocytes in the skin are regarded to be the key contributors or initiators of the disease. An increased production of TSLP by keratinocytes from atopic skin has been reported to further activate dendritic cells to drive Th2 polarization (Fig. 4)31.
Even though T cells which were previously described to be crucial for AD pathogenesis are dispensable under certain conditions and can be “replaced” by innate immune cells which include MCs, eosinophil’s and macrophages (Fig. 4)49-51. Likewise, AD can develop in the absence of IL-4, signal transducers and activators of transcription 6 (STAT6) and IgE, although the overexpression of IL-4 can trigger AD development in the skin52,53. Thus, AD seems to have highly superfluous mechanisms which converge furthermore with barrier impairment, xerosis and itch.
Findings showing that AD may be present in of two different immunological forms, the extrinsic AD (atopic eczema) and the intrinsic AD (non-atopic eczema)34,40 are underlining this complexity of AD. Generally, 20-30% of the patients are affected by intrinsic AD. These patients have no increased levels of allergen specific or total IgE nor eosinophil numbers; yet, the two subtypes are indistinguishable in their clinical presentation. Thus, based on the heterogeneity of AD, it is likely that immune deviations and aberrations in skin cells both can contribute to AD independently and set off its development54.
20
Figure. 4: Pathogenesis of atopic dermatitis.
In AD, barrier disruption leads to entry of antigens, which encounter langerhans cells, dendritic cells and activating Th2 cells. T cells produces IL-4 and IL-13 which stimulate keratinocytes to produce TSLP. Activated TSLP express OX40 ligand to induce Th2 cells. Cytokines and chemokines, such as IL-4, IL-5 and IL-13 produced by Th2 cells and DCs stimulate skin infiltration by inducing DCs, mast cells, and eosinophils.
Reprinted from Dhingra et al. 2013: Mechanisms of contact sensitization offer insights into the role of barrier defects vs. intrinsic immune abnormalities as drivers of atopic dermatitis, J Invest Dermatol.2311-4. (Oct 1, 2013.). Copyright (2014), with permission from Nature publishing group.
1.3. KERATINOCYTES
Keratinocytes are the highly specialized epithelial cells which maintain the physical and biochemical barrier integrity of the skin55,56. To form the skin barrier and to maintain the skin integrity, keratinocytes continuously undergo a complex differentiation process. The most relevant morphological and cytostructural changes of keratinocytes occur during differentiation in the spinous and granular layers.
21
During this process many different differentiation-dependent proteins are produced such as involucrin, filaggrin, transglutaminase, claudin etc.55. A dysregulation of these genes can lead to the skin disease and diminishment of skin barrier47,57-59. Studies have shown that cytokines produced by keratinocytes play a critical role in maintaining the immune response, cellular communication and in the pathogenesis of disease28,44,60. For the barrier function of the skin, cytokine signaling can result in multiple consequences e.g. proliferation and differentiation of keratinocytes which are influenced by cytokines production and are partly modulated by gene expression in these cells60. An increased expression of certain cytokines can result in an activation of complex network of signaling molecules which can disrupt the physiology of keratinocytes and the quality of the skin barrier3. Upon skin disruption, keratinocytes are stimulated and the production of different proinflammatory cytokines such as TSLP, TNF-α, IL-1α is initiated (Fig. 5)14.
1.3.1 Role of keratinocytes in skin irritation
As indicated above, keratinocytes are the most important cell type for maintaining the homeostasis of the skin. They provide a rigid structure by undergoing a differentiation process. During differentiation, numerous genes (e.g. loricrin, involucrin, pro-filaggrin etc.) are expressed and finally the cells enters into a cell cycle arrest61.
Keratinocytes are the main producers of many different inflammatory mediators during skin irritation. IL-1α is considered as one of the primary alarm signals followed upon skin disruption in the inflammatory cascade (Fig. 5)62. Several, in vitro studies have shown that different irritants are capable to induce IL-1α in keratinocytes61,63-65. The production of IL-1α further activates the release of other pro-inflammatory cytokines or chemokines such as IL-1β, TNF-α, IL-6, IL-8 by other epidermal and dermal cells66. IL-1β is produced in an inactive form by keratinocytes and cleaved into the active form by proteases which are not generally present in the resting keratinocytes. Proteases are activated upon irritation of keratinocytes with phorbol myristate acetate (PMA) or sodium lauryl sulphate (SLS)67. IL-1α along with 22
IL-1β has pleiotropic effects and is involved in the activation of dendritic cells and T cells67.
Figure 5: Role of keratinocytes in skin inflammation.
Skin barrier disruption allows microbes or irritant to enter in the skin which stimulates the keratinocytes and initiates the immune responses. Stimulated keratinocytes produces different proinflammatory cytokines such as TNF-α, TSLP, IL-1α etc. which leads to skin inflammation and further eczema development.
Adapted from Skin immune sentinels in health and disease. Frank O. Nestle, Paola Di Meglio, Jian-Zhong Qin and Brian J. Nickoloff, Nat Rev Immunol. Oct 2009; 9(10): 679–691.
1.3.2 Role of keratinocytes in AD
AD is characterized by itch and the onset of chronic or relapsing eczematous skin lesions68. A range of different factors and cell types are known to contribute to the pathogenesis of AD69. Keratinocytes are considered to be the primary source of barrier deficiency in AD development70. Since a decade, there has been better understanding in the role of keratinocytes in AD. Under AD environment, keratinocytes produces a unique pattern of cytokines and chemokine’s such as increased levels of chemokine ligand (CCL)5 (RANTES) after stimulation with TNF- α and IFN-γ71. It has been also shown that keratinocytes driven from AD patients produce more granulocytes- macrophage colony- stimulation factor and TNF-α 72.
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Other studies with stimulated keratinocytes of nonlesional skin from AD patients have shown a lower expression of beta-defensin-2, an antimicrobial peptide which chemoattracts Th17 cells compared to healthy or psoriasis controls73. More recent studies, showing the contribution of keratinocyte-derived cytokines such as TSLP on the inflammatory response provide a greater appreciation for the active role of keratinocytes not only as barriers to the environment74, but also as perpetuating cells with activating DCs to prime T cells to further produce IL-4 and IL-1371. TSLP activated DCs also produce chemokines such as CCL17 and macrophage derived CCL22, which further leads to the infiltration of Th2 cells in AD lesions38. Studies have also shown that activated keratinocytes produce IL-25 and IL-33 which than act on mast cells and antigen presenting cells (DCs and LCs)38,44.
1.4 TUMOR NECROSIS FACTOR-α (TNF-α) 1.4.1 TNF-α – a proinflammatory cytokine
Figure 6: Different forms of TNF-α.
Two forms of TNF-α present i.e. a) Soluble TNF-α (or secreted form) and b) Membrane TNF-α (or cell associated). Binding of TNF-α to its receptors TNFR1 and TNFR1 triggers intracellular signaling cascade. Upon activation, TNF receptor forms trimer which binds to the monomer of TNF-α which leads to the conformational change in to the structure of receptor.
Reprinted from Palladino et al. 2003: Anti-TNF-α therapies: the next generation: Nature Reviews Drug Discovery 2, 736-746 (September 2003). Copyright (2014), with permission from Nature publishing group.
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TNF-α was first identified as an endotoxin-induced glycoprotein which causes haemorrhagic necrosis of sarcomas in a mouse model. In 1984, the cDNA of TNF-α was first cloned and shown to have the structural and functional homology to lymphotoxin (LT) β and was described as (LT) α75,76. TNF proteins are ubiquitously expressed by different cell types of the innate and acquired immunity such as B cells, T cells, NK cells, DCs, and monocytes3. TNF-α is expressed in two different forms, one is the cell-associated or membrane TNF-α (26-kDa) and the other one is the secreted or soluble TNF-α (17-kDa) form 77(Fig. 6). Both forms of TNF-α are biologically active. The cell-membrane bound form of TNF-α is thought to be responsible for juxtacrine signalling whereas secreted form for the direct cell-to-cell contact, though the exact functions of these two forms are still controversial 77,78. Based on numerous studies, TNF-α is considered as one of the best known proinflammatory cytokine having a crucial role in host defense and inflammatory diseases79,80. It has been associated with the development of many autoimmune disorders such as rheumatoid arthritis, psoriatic arthritis and inflammatory bowel disease77. TNF-α is also known to enhance disease severity by its capability to induce different proinflammatory cytokines, such as IL-1 and different chemokines81. The administration of TNF-α antibodies and its interference with the TNF pathway are widely used for controlling pathogenesis of many diseases such as rheumatoid arthritis, psoriasis, inflammatory bowel disease 77,81. Since the last 10 years, monoclonal antibodies against TNF-α or its receptor are widely used in the clinic for the blockage of TNF pathway81 for the treatment of autoimmune diseases like rheumatoid arthritis, but also psoriasis.
1.4.2 Role of TNF-α in skin irritation
The exposure of the skin to various irritants or chemicals results in skin irritation. Skin irritation is a complex process which involves a series of responses such as skin damage, cell death and activation of keratinocytes and other cells82. Keratinocytes are well known to produce large amounts of proinflammatory cytokines such as TNF- α, IL-1β, IL-6 (Fig. 5)14. The upregulation of TNF-α in the skin during irritation has been shown by different irritants e.g. dimethyl sulfoxide, PMA, formaldehyde,
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tributyltin, and SLS67. TNF-α has pleiotropic effects on keratinocytes and endothelial cells, where it increases the expression of major histocompatibility complex class II molecules and upregulates cell adhesion molecules e.g ICAM-1. TNF-α is also capable of inducing inflammatory factors such as IL-1, IL-6, IFN-γ, granulocyte- macrophage colony-stimulating factor (GM-CSF) and CXC ligand 8 (CXCL8)56. During irritation, TNF-α has common functions with IL-1α as a primary alarm signal to other cell types, to further initiate the release of CCL20 and CXCL8 chemokines production from macrophages. An increased expression level of CCL20 and CXCL8 leads to the migration of cells to the site of injury. T-cells, but also immature DCs are activated83. The important role of IL-1α and TNF-α in the pathogenesis of skin irritation has been proven at genetic levels. It has been shown, that certain genetic polymorphisms of both TNF-α and IL-α are linked with an altered risk of skin irritation.
Individuals with TNFA-308 polymorphisms have a lower risk to develop ICD whereas TNFA-238 alleles have an increased risk to ICD. Likewise, IL1A-889 C/T alleles are protective for the development of ICD, clearly indicating that these genetic polymorphisms are associated with an increased or decreased risk of ICD development67. Hanel et al 2013 have shown the involvement of TNF-α in barrier repair. TNF-α inhibited the expression of skin barrier genes such as filaggrin and loricrin, TNF-α thereby weakening the skin barrier3. The central role of TNF-α in skin irritation was further confirmed by the direct administration of TNF neutralizing antibodies in vivo. These studies show, that the skin inflammation was reduced upon antibody administration84,85.
1.4.3 Role of TNF-α in AD
The direct role of TNF-α for the development of AD is not completely understood. A detailed analysis of the literature revealed a negative association between TNF and AD development86-89. The most remarkable evidence for a functionally relevant inverse association between TNF and AD comes from different clinical studies, which have reported the onset of possible AD as a side effect upon anti-TNF therapy in single patients with rheumatoid arthritis, Crohn's disease and psoriasis 90,91. On the other hand few reports show a beneficial effect of TNF-α directed therapy in single 26
AD patients92. These patients suffered from AD subsets (long-lasting and/or combined with contact dermatitis). Another evidence of defective TNF production in AD patients came from an analysis of peripheral blood leukocytes, in which decreased TNF-α production was consistently reported in AD patients87-89. Recent studies indicated that cytokines like IL-1β, IL-4, IL-5, IL-12, and IFN-γ are enhanced, whereas TNF-α levels are reduced in AD skin compared to healthy controls88. Although TNF-α is undoubtedly one of the best-characterized proinflammatory cytokines, it can also exert anti-inflammatory effects and contribute to the resolution of inflammatory diseases by various mechanisms, e.g. by promoting cluster of differentiation (CD) 4+CD25+ T regulatory cells93, by mediating apoptosis of auto- reactive effector T cells94 and by inducing local glucocorticoid production95.
1.5 THYMIC STROMAL LYMPHOPOIETIN (TSLP)
TSLP is an IL-7 like cytokine and has been first discovered in the culture supernatants of mouse thymic stromal cells which gave rise for this nomenclature.
TSLP supports the growth and differentiation of B cells but also the proliferation of T cells96,97. Different groups throughout the world demonstrated that high affinity TSLP binding requires the combined binding to the IL-7 receptor α-chain and TSLP receptor (TSLPR)97-99. TSLP is mainly expressed by epithelial cells from the thymus, the skin, the lung, the intestine and tonsils as well as by stromal cells and mast cells100-103. In the thymus, TSLP is responsible for the differentiation of Treg cells by instructing thymic DCs104. Interestingly, human TSLP does not exert the same functions as its murine counterparts; however it does activate immature CD11c+
myeloid DCs101,103. Thus, DCs can activate naïve CD4+ T cell proliferation and initiate the production of IL-4, IL-5, IL-13 and TNF-α (Fig. 7). In contrast, the production of the anti-inflammatory cytokines IL-10 and IFN-γ is inhibited by TSLP- induced DCs103. TSLP is known to activate the upstream component of JAK1 and JAK2, which bind to IL-7Rα and TSLPR chain8. Subsequently JAK1/2 are phosphorylated and activate STAT5105. TSLP binding may also lead to an activation of the subsequent STAT family members 1, 3, 4 and 6106,107. Recent 27
phosphoproteomic data show that TSLP is also involved in a number of additional signalling pathways. It was shown that often signal transduction like Erk1/2, JNK1/2and p38 were phosphorylated after TSLP dependent activation108. TSLP exerts its effects on a broad range of cells. Therefore it has been implicated to play an important role in many diseases like infections, cancer and inflammatory bowel diseases109-111. However, an even more important role of TSLP has been anticipated in allergic diseases like AD and asthma112. TSLP has been shown to be upregulated in an OVA-driven mouse model of airway inflammation113. These observations were confirmed in an OVA-induced murine model of allergic asthma and AD with TSLPR-/- mice which show a defective airway inflammation and allergic skin inflammation114,115.
1.5.1 Role of TSLP in skin irritation
An acute insult against the stratum corneum results in perturbation of the barrier integrity and induces a process of positive and negative alarm signals which initiate both homeostatic and proinflammatory responses in the skin22,116. The compromised barrier integrity further triggers the production of critical cytokines to initiate skin inflammation117-119. TSLP is one of the cytokines which is expressed by keratinocytes in response to physical injury and inflammatory cytokine stimulation (Fig. 7)74. The crucial role of TSLP in allergic inflammation is well established but the underlying mechanisms behind the trigger of TSLP production by different factors are still unknown50,120,121. Primary human keratinocytes and skin explants were shown to produce TSLP upon bacterial, viral or inflammatory stimuli or physical trauma 122,123. Angelova-Fischer et al. (2010) investigated the role of tape stripping and SLS on skin irritation and show that the stratum corneum of the epidermis is damaged, which is associated with an increased TSLP expression117. They also observed that keratinocytes express TSLP in the suprabasal cell layers of the epidermis. Among these layers it is mainly localised in the granular and spinous
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layer and is not expressed by keratinocytes in the basal layer. These data are in alignment with previous observations which have shown that TSLP expression is a characteristic sign of keratinocytes which are undergoing a differentiation process103,124. As previously described, human TSLP can induce synergistic effects between proinflammatory and Th2 cytokines123. On the other hand keratinocytes from Notch-deficient mice show an increased level of TSLP expression and an eczema-like phenotype in skin upon barrier disruption 123,125,126 indicating that there is a link between barrier integrity and TSLP production.
Figure 7: TSLP induction in keratinocytes.
Skin barrier disruption, allergen or Th2 derived cytokines triggers the epithelium cells for TSLP production.
TSLP activates DCs for the further recruitment of T cells for further production of proinflammatory cytokines or chemokine’s such as IL-4, IL-5, and TNF-α. TSLP also activates mast cells to produce other cytokines e.g. IL- 13, IL-5 and TSLP itself (not shown).
Reprinted from Hamida Hammad et al. 2008: DCs and epithelial cells: linking innate and adaptive immunity in asthma: Nature Reviews Immunology 8, 193-204 (March 2008), Copyright © 2008, with permission from Nature Publishing Group (2014).
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1.5.2 Role of TSLP in AD
Many factors can elicit AD when overexpressed, though not being absolutely essential. The role of TSLP in AD development was not clear until studies showed that an overexpression of TSLP in the skin of mice leads to the development of a
“spontaneous” dermatitis, the most characteristics feature of human AD49,103. Since TSLP is primarily produced by epithelial cells, this provided further evidence to the theory of KCs as the “initiators” of AD (Fig. 7)127. Later on various groups confirmed TSLP as a major initiator of AD50,51,128. Another study has shown that a direct administration of TSLP into the skin leads to AD-like lesions74. Although this thesis is focusing on the skin, similar results were obtained for atopic asthma models60,129. TSLP is involved in the proliferation and differentiation of Th2 cells and the subsequent production of IL-4, IL-5, IL-13 and TNF-α103. Moreover, it was found that TSLP is highly expressed in keratinocytes from AD patients with acute and chronic lesions. Additionally it is associated with the activation and migration of DCs within the dermis103. Therefore, TSLP was suspected to be one of the initiating factors for the development of AD.
Yoo et al. (2005) reported that keratinocyte specific overexpression of TSLP elicited skin disease with all the characteristic features of human AD, such as edema hyperkeratosisa, dermal mononuclear cell infiltrate49. Mice lacking T cells, but overexpressing keratinocyte-specific TSLP still develop skin inflammation, indicating that T cells are not required for disease progression49. Other studies with different AD models using TSLPR-/- mice show that TSLP is necessary to induce AD i.e.
TSLP-/- mice failed to develop AD115,130.
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1.6 OBJECTIVES
Over the years, TNF-α have been well characterized as crucial proinflammatory cytokine with its roles in both host defense and inflammatory diseases80. Consequently, anti-TNF therapies are an approved treatment for autoimmune diseases, including rheumatoid arthritis and psoriasis77 with eczema development as the most common side effect90,91. However the role of endogenous TNF-α in acute skin irritation and in AD development is not well understood. In this thesis, the role of endogenous TNF in skin irritation but also in an AD model was analyzed in TNF-α deficient mice.
Within this thesis the following questions were addressed
1) Can the clinical observations be replicated in a murine disease model? And if so, what are the mechanisms?
2) Is irritation responsible for TSLP induction outside of a typically allergic condition, and what are the associated mechanisms?
3) Is TSLP is the factor responsible for the exaggerated dermatitis in the absence of TNF?
3) Are TNF-/- mice inherently prone to increased TSLP production or does it require the micromilieu of the AD?
5) Does TNF deficiency lead to enhanced TSLP production through an indirect mechanism by affecting the micromilieu and whether and to what extent are MCs crucial elements in this cascade?
To answer these questions will open a novel view on the inflammatory processes operating in the initiation and development of AD.
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2. MATERIAL AND METHODS 2.1 MATERIALS
Details about antibodies, instruments, chemicals, buffers, solutions, reagents, labwares and software used are listed below:
Table 1: List of reagents
Reagent Supplier Catalog
Number
α-monothioglycerol Sigma-Aldrich M-6145
Agarose Biozym 840004
Albumin from chicken egg white (OVA) Sigma-Aldrich A5503-10G
Anti IgE BD Pharmingen™ 553413
Antibody diluent (Dako REALTM) DAKO Diagnostika S0809
Aqua Braun 2351744
Avidin/Biotin Blocking Kit Vector Laboratories,
Inc. SP-2001
Bovine serum albumin (BSA) PAA K45-001
Calcitriol Sigma-Aldrich D1530
Croton oil Sigma-Aldrich C6719
DermaLife K Medium Complete Kit Lifeline Cell
Technology LL-0007
Dispase BD Biosciences 354235
Desoxyribonucleic acid (DNA) Molecuar
Weight XIII – 50 base pair (bp) ladder Roche 11721925001 DNA Molecular Weight XIV – 100 bp ladder Roche 11721933001
En Vision+ System-HRP(AEC) Dako K-4005
Ethanol J.T. Baker 8025
Ethidium Bromide Solution Invitrogen 15585-011
Fetal Bovine Serum (FBS) PAA NC9862466
IgE BD Pharmingen™ 554118
IMDM medium PAA E-15-819
Hydrogen peroxide (H2O2) Sigma-Aldrich 216763
Histamine Sigma-Alrich H7125
Human TSLP ELISA kit eBioscience 88-7497-88
LightCycler® FastStart DNA Master SYBR
Green I Roche 12239264001
LSAB2 System-HRP Dako K0675
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Mouse TSLP Duo Set R&D Systems® DY555
Nafamostat mesylate Sigma-Aldrich N-0289
Nucleo Spin® RNA II Macherey-Nagel 740955.250
PBS GE Healthcare H15-002
Penicillin/Streptomycin Biochrom A 2212
Peroxidase block Dako S2001
Phorbol 12-myristate 13- acetate(PMA) Sigma-Aldrich P 8139
Proteinase K Macherey-Nagel 740506
Recombinant Mouse Mast Cell Protease-
6/Mcpt6 R&D Systems® 3736-SE-010
rh Skin beta Tryptase Promega G7061
Retinoic Acid Sigma-Aldrich R4643
rhIL-1β Immunotools 11340015
rhTNF-α Immunotools 11343013
rm IL-4 Peprotech 11340043
rmIL-1β Miltenyi 130-094-053
rmTNF-α Miltenyi 130-094-085
rm IL-25 eBioscience 14-8175-62
rm IL-3 Immunotools 12340033
rm IL-33 eBioscience 14-8332-62
rm IL-4 R&D 404-ML-010
Sodium dodecyl sulphate(SDS) Sigma-Aldrich L3371
TAE buffer (50x) Genaxxon M3087.1000
Tetramethylbenzidine Sigma-Aldrich T5525
TLR3 ligand InvivoGen tlrl-pic
Transcriptor High Fidelity cDNA Synthesis
Kit Roche 05081963001
Trypsin / EDTA Solution Gibco® BD R-001-100
Trypsin inhibitor from Glycine max
(soybean) Sigma-Aldrich 9035/81/8
Tween 20 Sigma-Aldrich P1379-500ML
Xylol Roth 9713.3
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Table 2: List of antibodies and antagonist
Antibody Supplier Catalog
Number
Anti-mouse TSLP R&D Systems® AF555
Biotin-sp-conjugated affinipure F(ab’)2
fragment rabbit anti goat IgG(H+L) Jackson
immunoresearch 305-066- 003 Fluorescein iso thiocyanate
(FITC) conjugated αm CD117 (c-kit), Clone 2B8
eBiosciences 11-1171-82
Purified NA/LE Rat Anti-Mouse CD117 BD Pharmingen™ 553867 Purified NA/LE Rat IgG2b, κ Isotype
Control BD Pharmingen™ 556968
PE conjugated αm FceRI α, clone: MAR-1 eBiosciences 12-5898-81
Mouse IgG2a R&D Systems® MAB003
Mouse mast cell protease-6/Mcpt6
antibody R&D Systems® AF3736
Mouse TSLP Antibody R&D Systems® MAB555
Mouse IgG2A Antibody R&D Systems® MAB003
Rabbit anti-human IL-1α antibody Abcam ab9614
Rabbit anti-mouse IL-1α antibody Abcam ab9724
Rabbit IgG Abcam ab27478
rmIL-1Ra Immunotools. 12344870
rhIL-1Ra Immunotools. 11344874
Table 3: List of materials
Material Supplier Catalog
Number Biosphere® Filter Tips
0.5-20 µL 2-100 µL 100-1000 µL
Sarstedt
70.1116.210 70.760.212 70.762.211
Cell strainer, 40 µm BD FalconTM 352340
Cell strainer ,100 µm BD FalconTM 352360
Culture flask T 75 T 175
Cellstar®, Greiner-Bio
658175 660175
Conical tube ,15 mL BD FalconTM 352096
Conical tube ,50 mL BD FalconTM 352070
Descosept AF Dr Schumacher GmbH sc 311001
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LightCycler® Capillaries Roche 04929292001
Micro tube, 0.5 mL Sarstedt 72.699
Micro tube, 1.5 mL Sarstedt 72.690.001
Micro tube, 2 mL Sarstedt 72.691
Precellys Steel Kit 2.8 mm Peqlab 91-PCS-
MK28 Quality Tips without filter
10 µL 200 µL 1000 µL
Sarstedt
70.1130 70.760.002 70.762 Serological Pipet
5 mL 10 mL 25 mL
BD FalconTM
357543 357551 357525 96-well cell culture plate Cellstar®, Greiner-Bio 655185
Petri dish Greiner-Bio 632181
Table 4: List of instruments
Instrument Type Supplier
Cell counter CASY® - TTC-2FC-1142 Innovatis AG, Reutlingen
Centrifuge Megafuge 1.0R Thermo Scientific,
Schwerte
CO2-Incubater HERAcell® Thermo Scientific,
Schwerte
Electrophoresis System Sub-Cell® GT Bio Rad, München
Gel Imager Gene Genius Syngene, Cambridge
Inverted Reflected-Light Microscope Zeiss Axiovert 10 Zeiss, Jena
Light Cycler Roche,Penz
berg
Flow Cytometer MACS Quant Miltenyi Biotec,
Bergisch Gladbach
Microplate reader Dynatech MRX Dynex
Technoloies, Chantilly
Multipipette Multipipette® plus Eppendorf, Hamburg
Pipette Eppendorf Reference® /
Research® Eppendorf, Hamburg
Pipettor Pipetus standard Hirschmann
Laborgeräte,
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Instrument Type Supplier Eberstadt
PCR machine Px2 Thermal Cycler Thermo Electron
Corporation
Power Supply POWER PAC 300 BioRad, ‚München
Spectrophotometer Nano Drop 1000 Thermo Scientific, Schwerte
Tabletop centrifuge with refrigeration Centrifuge 5417C Eppendorf, Hamburg
Tabletop Centrifuge Centrifuge 5417R Eppendorf,
Hamburg
Thermomixer Thermomixer comfort Eppendorf,
Hamburg
Tissue homogenizer Precellys 24 Bertin Technologies,
Montigny-le- Bretonneux
Waterbath MA6 Lauda, Lauda-
Königshofen
Vortexer REAX 2000 Heidolph,
Schwabach
2.2 METHODS
2.2.1 Animal experiments
2.2.1.1 Breeding of B6;129S-Tnftm1Gkl/J (TNF-/-) mice
TNF-/- mice were provided by Professor Max Löhning from DRFZ, Berlin. To generate these mice, targeting vector was constructed by replacing TNF gene with MC1neopA cassette (Stratagene) the 438 bp Narl-BglII fragment containing 40 bp of the 5' UTR, all the coding region, including the ATG translation initiation codon, of the first exon and part of the first intron of the mTNF-α gene131. These mice were bred and maintained under pathogen free conditions in animal facility. All experiments were performed according to German animal protection law.
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2.2.1.2 Genotyping of TNF-/- mice
Genomic DNA was isolated from 5 mm2 tail biopsies of TNF-/- mice by using the nucleospin tissue kit, according to manufacturer’s protocol. PCR was performed to identify the genotype of mice. TNF-α gene primer sequences were obtained from the ‘The Jackson laboratory’ site (strain stock no.: 003008) and were synthesized from TIB MOLBIOL, Berlin, Germany and are specified below:
Primer Sequence:
Primer Sequence Primer type (short name) oIMR4182 5’-tagccaggagggagaacaga-3’ Common (GC)
oIMR4183 5’-agtgcctcttctgccagttc-3’ Wild type Reverse (GW) oIMR7297 5’-cgttggctacccgtgatatt-3’ Mutant Reverse (GM)
Reaction component:
Regents Volume (µl) Final concentration
10x GenTherm buffer 1.2 1x
50 mM MgCl2 0.48 2 mM
10 mM deoxyNTPs 0.24 200 nM
10 μM forward primer (GC) 1.2 1 μM
10 μM reverse primer (GW) 1.2 1 μM
10 μM reverse primer (GM) 1.2 1 μM
50 U/μl DNA polymerase 0.075 0.03 U/μl
DNA 2
dH2O (makeup the volume up to 14µl)
The following PCR program was used:
94 °C - 3 min 94 °C - 30 sec
62 °C - 1 min 35 cycles 72 °C - 1 min
72 °C - 2 min
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