mechanisms underlying the regulatory function of tumor

Mechanisms underlying the regulatory function of tumor necrosis factor-α in skin inflammation

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

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

Table of contents

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

Table of contents

4.5 CONCLUSION AND OUTLOOK .................................................................................... 84

REFERENCES ............................................................................................................ 87

APPENDIX .................................................................................................................. 99

ACKNOWLEDGEMENTS ......................................................................................... 101

SELBSTÄNDIGKEITSERKLÄRUNG / DECLARATION .......................................... 103

5

List of abbreviations

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

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

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

Introduction

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

Introduction

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

Introduction

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

Introduction

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

Introduction

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

Introduction

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

Introduction

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

Introduction

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

Introduction

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

Introduction

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

Introduction

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

Introduction

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.

23

Introduction

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.

24

Introduction

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,

25

Introduction

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

Introduction

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

Introduction

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

28

Introduction

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).

29

http://www.nature.com/nri/journal/v8/n3/full/nri2275.html

http://www.nature.com/nri/journal/v8/n3/full/nri2275.html

Introduction

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.

30

Introduction

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.

31

Material and methods

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

32


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

33


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

34


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,Penzberg

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,

35


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.

36


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

37


4 °C - onhold

2 μl of 10x DNA loading dye were added to each PCR products and separated on a

2 % agarose gel. Gels were photographed with a UV light photometer and bands

were further analysed to determine the genotype of the mice.

Expected band: Mice Band size

TNF-/- homozygous 318 bp

TNF-/- heterozygous 183 bp and 318 bp

Wildtype (wt) 183 bp

2.2.1.3 In vivo skin irritation model

Figure 8: Experimental scheme of skin irritation model with different irritants treatment in vivo.

10 week old female C57BL/6 (wt) and TNF-/- mice were gently dry shaved at three

different regions and exposed 30 times either to croton oil, 1% SDS or tape

stripping using cotton swabs or cello tape. The fourth skin region was shaved 30

times with a help of wet shaver (Fig. 8). The groups of mice were sacrificed after 4

38


and 18 hr, and blood was collected for serum. 5 mm2 skin biopsies were collected

for immunochemistry and mRNA isolation.

2.2.1.4 Mouse model of AD

Figure 9: Experimental scheme of mouse model of AD with different antibodies/cytokines treatment in vivo

To induce AD, an adapted protocol from Dahten et al 2008 was used132. Briefly, 10

weeks old female wt and TNF-/- mice were sensitized by three subsequent

intraperitoneal injections (i.p) with 100 μl of 10 μg ovalbumin (OVA) adsorbed to 1.5

mg Al(OH)3 (alum) on days 1, 14 and 21 (black arrows in Fig. 9). On day 21, the

belly of the mice was shaved by wet shaving, further tape stripped and 100 µg OVA

allergen was applied epicutaneously by the patch test method for one week period.

Each mouse had a total of three one week allergen exposures at the same site on

the skin with a two week intervals in between without any allergen.

To better understand the intrinsic role of TNF in skin inflammation, different

mediators and specific antibodies were applied intradermal (i.d) to the mouse skin

one day before the patch, half a day before the patch renewal and in the middle of

the patch-free week (blue arrows in Fig. 9). The dose and timing schedule of the

antibody application was based on data from the literature i.e. anti-TSLP 20

µg/mice i.d.133, anti-c-Kit 40 µg/mice i.d134.

On day 71, mice were anesthetised by isoflurane and sacrificed by cervical

dislocation. Blood was collected for further analysis of immunoglobulin and

cytokines levels in the serum. Photographs of the patch area were taken for the

39


assessment of the symptom score. 5 mm2 skin biopsies from lesional skin were

taken for immunohistochemistry in O.C.T compound and frozen slowly into liquid

nitrogen or in formalin for paraffin embedding. Rest of the skin was frozen into liquid

nitrogen for mRNA isolation. All frozen samples were stored at -80 °C until further

analysis.

2.2.1.5 Assessment of AD symptoms

AD severity was evaluated by using a skin score which nearly resemble to a score

which is widely used in clinical practice. The SCORAD (scoring of atopic dermatitis)

considers different clinical features to determine the severity of AD in humans135. In

our model such typical features used to evaluate the severity were papulation,

erythema, excoriation/crusting, dryness and extension of the lesions. Each

parameter was evaluated independently in a blinded manner by six individuals in a

randomized order. Severity for each parameter was rated as following: 0, no

symptom; 1, mild symptoms; 2, intermediate symptoms; and 3, severe symptoms.

The score from all the six individuals for each of these factors were then summed

up together and the total skin score was taken as AD severity with maximal skin

score considered as 15 and minimal 5.

Functional skin barrier assessment AD severity was further evaluated at a functional level by measuring TEWL in the

skin. This method measures the barrier dysfunction which is developing in

eczematous skin. During the measurement, the probe was placed on the belly of

the mice to measure TEWL. The vapor gradient density was measured indirectly by

two pairs of sensors i.e. sensors of temperature and relative humidity inside the

hollow cylinder of defined volume and analyzed by a microprocessor. The

measurement of TEWL is based on diffusion principle in an open chamber TEWL

machine136.

Blood samples Blood samples were taken on days 0 and 35 from the vena facialis with a micro

lancet by punction. On day 71, complete blood was withdrawn from retro orbital

40


venous sinus located behind the eyes. The blood was collected into special serum

separator tubes and centrifuged at 14,000 rpm for 10 min. Serum was further stored

at -80 °C until further analysis.

2.2.2 Cell culture methods

2.2.2.1 In vitro culturing in mouse and human

2.2.2.1.1 Isolation, culturing and treatment of primary Keratinocytes

Figure 10: Example of murine keratinocyte culture. Keratinocytes were isolated from murine skin and cultured. A) Shows first growing colonies of freshly isolated

keratinocytes and B) shows the confluent cells ready for passage. (385 x magnification).

Mouse keratinocytes: Mice were anesthetised by isoflurane and sacrificed by cervical dislocation. The

skin was gently shaved and the primary keratinocytes were isolated according to a

published protocol with few adaptations137. Cells were cultured in DermaLife® K

serum-free keratinocyte culture medium supplemented with essential factors, 30 µM

calcium chloride and penicillin/streptomycin (KC medium)(Lifeline Cell Technology,

Walkersville, MD, USA).

After the KCs reached 70-80% confluency, cells were passaged using trypsin-

Ethylenediaminetetraacetic acid (EDTA) (PAA Laboratories, Cölbe, Germany). Cells

were counted and cell viability was checked by CASY® Cell Counter (CASY) or by

trypane blue using haemocytometer. After the 2nd passage, 7.5x103 cells per well

were seeded in a 96-well plate to grow for 96 hr in hydrocortisone hemisuccinate 41


free KC medium. Cells were stimulated with 10 μg/ml TLR3-ligand, 20 ng/ml rmIL-

1β, 20 ng/ml rmTNF-α, 20 ng/ml rmIL-4, 10 ng/ml rmIL-25, 50 ng/ml rmIL-33 or 50

ng/ml PMA for 24 hr. Supernatants were collected and measured by a mouse TSLP

enzyme linked immunosorbent assay (ELISA) Kit. (R&D Systems, Minneapolis, MN,

USA).

Human keratinocytes: Human KCs were isolated from foreskin and processed as previously described138.

The skin was obtained after circumcisions, with informed consent of the patients

and approval by the university Ethics committee. All the experiments were

conducted according to the Declaration of Helsinki Principles. After the 2nd passage,

7.5x103 cells/well were seeded in a 96-well plate in KC medium and grown to 70-

80% confluence. After reaching confluence, the medium was changed to

hydrocortisone hemisuccinate free KC medium for 24 hr, and cells were stimulated

with 10 μg/ml TLR3-ligand, 20 ng/ml rhIL-1β, 50 ng/ml rhTNF-α and 20 ng/ml rhIL-4

for 24 hr. Supernatants were collected and measured by a human TSLP ELISA Kit.

(R&D Systems, Minneapolis, MN, USA).

2.2.2.2 Ex vivo culture and stimulations Mice were anesthetized and sacrificed by cervical dislocation. Skin of the mice was

gently shaved and 5 mm2 of biopsy punches were taken from the dissected skin.

The initial protocol was adopted from as previously described139. After the skin

biopsies were treated by 1% SDS, croton oil by the aid of a cotton swab and

physical scratching by a scalpel for 30 times each, biopsies were incubated in 150

μl of KCs medium without hydrocortisone hemisuccinate for 8 hr. Skin biopsies

were also stimulated with 20 ng/ml rmIL-1β, 20 ng/ml rmTNF-α and 20 ng/ml rmIL-4

for 8 hr. After stimulation, supernatants were collected and TSLP was quantified by

ELISA.

Inhibition experiments (mouse): 5 mm2 skin biopsies were immersed in to 1% SDS for 5 min followed by 5 times

washing. After washing, biopsies were stimulated with 200 ng/ml of rmIL-1Ra, 25

ng/ml of neutralizing αm IL-1α antibody and 25 ng/ml rabbit-IgG in to 150 µl of KCs 42


medium without hydrocortisone for 3 hr. After 3 hr, supernatants were collected and

TSLP was quantified by ELISA.

Inhibition experiments (human): Epidermal sheet from foreskin were isolated with overnight treatment with dispase

II. 5 mm2 of small pieces of epidermal sheet were cut carefully and immersed in 1%

SDS for 3 min, followed with 5 times extensive washing with KCs medium and

treated with 200 ng/ml of rhIL-1Ra, 1 µg/ml of neutralizing anti-human (αh) IL-1α-

antibody (and its respective concentration of rabbit-IgG as control) for 3 hr in KCs

medium without hydrocortisone. After stimulation, supernatant was collected and

TSLP ELISA was performed.

2.2.3 TSLP enzyme linked immunosorbent assay (ELISA)

Figure 11: Scheme of sandwich based - enzyme linked immunosorbent assay (ELISA) (adapted from Epitomics - an Abcam Company).

ELISA is an enzyme immunoassay used to measure the unknown level of antigens

in serum or supernatant. In this study we have used sandwich based ELISA to

quantify the level of protein. Here, first the primary antibodies were coated on the

surface of the plate and the target protein from serum or supernatant were

incubated for specific binding. The detection antibodies were incubated over the

surface of bound specific antigen. In the next step, the plates were incubated with

Horseradish peroxidase (HRP) linked biotinylated antibodies, which can convert a

chromogenic substrate. The enzymatic reaction leads to the color change which

was measured by spectrophotometer. The concentration of protein in the samples

was calculated by the means of standard curve. All the steps were performed at

room temperature and in dark from HRP-linked antibody.

43


Mouse and human TSLP ELISA: In vitro, ex vivo, or in vivo experiments were performed and cell free supernatant or

serum from mice and human epidermal sheet were obtained and measured for

mouse and human TSLP levels. Analysis was performed based on TSLP ELISA kit

from R&D system (mouse) and ebiosciences (human) according to manufacturer’s

instructions.

2.2.4 RNA isolation

Frozen skin samples from mice were homogenized by pre-chilled precellys

homogenisation (PEQLAB, Germany) in 500 μl RA1 buffer (NucleoSpin® RNA

isolation kit) along with 5 μl β-mercaptoethanol (β-Me) at 5500 rpm for 2*30 sec with

5 sec pause. Homogenized samples were transferred to NucleoSpin filter and

centrifuged at 11,000 g for 2 min at room temperature. Supernatant was taken out

carefully without disturbing the pellet and 500 μl of RNase-free water was added

along with 10% proteinase K and mixed well for tissue digestion. The lysate was

incubated for 15 min at 55 °C. After 15 min, lysate was spun down at 10,000 g for 3

min. Further, RNA isolation was performed according to manufacturer’s instruction

along with DNase digestion step for 15 min at room temperature. RNA was eluted

with 60 μl of RNase-free water. Using NanoDrop UV-Vis spectrophotometer, RNA

concentration was measured at 260 nm. Later, quality of RNA was checked by 2%

agarose gel. The eluted samples were stored at -80 °C for further analysis.

2.2.5 Reverse transcription

Total RNA was reverse transcribed into single stranded cDNA with TaqMan®

reverse transcription reagent according to manufacturer instructions. The kit

contains a recombinant Moloney Murine Leukemia Virus Reverse Transcriptase,

random hexamers and oligo d(T). 1 µg of total RNA was used for reverse

transcribtion in to cDNA in thermo cycler with following protocol.

44


Steps Temperature (°C) Time (min)

Incubation 25 10

Reverse transcription (RT) 48 40

RT inactivation 95 5

All cDNA samples were stored at -20 °C.

2.2.6 Real-time polymerase chain reaction

After RNA was reverse transcribed into cDNA with TaqMan reverse transcription kit

(Applied Biosystems, Darmstadt, Germany), fluorescence based real time

quantitative polymerase chain reaction (qPCR) was performed for the quantification

of gene expression in skin samples. qPCR was performed with LightCycler®

FastStart DNA Master SYBR Green I (Roche) according to the experimental

protocol below. The cDNA was pre-diluted 1:3 and the primers used were designed

by Primer3 software and are listed below. The formation of PCR product is

measured by increased level of fluorescence caused by specific binding of SYBR

green fluorescence dye to double-stranded DNA (SYBR green- Double-

Stranded DNA (dsDNA)). To ignore the non-specific binding by SYBR green, PCR

buffer also contains a reference dye to normalize the specific binding. The cycle

number of crossing point (CP) or the threshold cycle value (CT) is the number of

cycle at which significant increase of the normalized florescence is first measured.

Depending on CT values of a gene and the efficiency of primers, the relative

expression of a gene was calculated. The expression level of target gene was

normalized to the expression level of housekeeping gene i.e hypoxanthine-guanine

phosphoribosyltransferase (HPRT) using the 2-ΔΔCT method140.

45


Reagent Volume/sample (µl) Final concentration

10X FastStart DNA Master SYBR

Green I

0.50 1X

25mM MgCl2 0.80 3-5 mM

10µM Forward Primer 0.25 100-500 nM

10µM Forward Primer 0.25 100-500 nM

RNase-free H2O (makeup the volume up

to 3µl)

cDNA 2 (1:3 diluted stock)

Primer Sequence:

Gene

Primers

Sequence

Size

Product size

mHPRT

forward

reverse

5’-cgtcgtgattagcgatgatg-3’

5’-aatccagcaggtcagcaaag-3’

20

20

221

mDef B2 forward

reverse

5’-cactccagctgttggaagttt-3’

5’-gcaacaggggttcttctctg-3’

20

20

148

mDef B3 forward

reverse

5’-ctccacctgcagcttttagc-3’

5’-ggaactccacaactgccaat-3’

20

20

118

mIL-1α forward

reverse

5’-gctgaaggagttgccagaaa-3’

5’-cccgactttgttctttggtg-3’

20

20

146

mIL-1β forward

reverse

5’-tgaaatgccaccttttgaca-3’

5’-cttctccacagccacaatga-3’

20

20

190

mIL-4 forward

reverse

5’-gactctttcgggcttttcg-3’

5’-tgatgctctttaggctttcca-3’

19

21

105

mIL10

forward

reverse

5’-tttaagggttacttgggttgc-3’

5’-agggtcttcagcttctcacc-3’

21

20

137

mIL-33 forward

reverse

5’-atgggaagaagctgatggtg-3’

5’-ccgaggactttttgtgaagg-3’

20

20

150

mIFN-γ forward

reverse

5’-aactattttaactcaagtggcatagat-3’

5’-tgctgttgctgaagaaggtag-3’

27

21

217

mTslp forward

reverse

5’-agagaagccctcaatgacca-3’

5’-ggacttcttgtgccatttcc-3’

20

20

82

46


2.2.7 Isolation and culture of bone marrow cells and generation of bone marrow-derived mast cells (BMcMCs)

To isolate and culture bone marrow-derived mast cells, initial protocol was adopted

by Mrabet-Dahbi et al. 2009141. 10 week old wt and TNF-/- mice were sacrificed by

cervical dislocation. Skin was dissected and legs were separated from the hip to

foot. Foot was removed by cutting off the skin and ligaments. Muscles and tissue

from the leg was completely removed and tibia was separated from femur bone

without breaking the bones. Both bones were cleaned by Softasept®N and placed

into the falcon with washing medium (IMDM medium + 10% of

penicillin/streptomycin). Under sterile condition, bones were cut from both the side

and flushed with 10 ml of syringe filled with washing medium into a petri dish. After

all the bones were flushed out, single cell suspension was made by pipetting the

cells up and down. Cells were centrifuged at 1200 rpm for 10 min at 4 ºC. The

medium was discarded and cells were resuspended in to 20 ml of culture medium

with 10 ng/ml of IL-3 for mast cell differentiation. On day 5 and 8 cells were

centrifuged at 1200 rpm for 10 min at 4 ºC and further resuspended in to fresh

medium along with 10 ng/ml IL-3 and placed in to a new flask. On day 15, the

medium was changed and cells were moved in to a big flask with 40 ml of culture

medium. On day 19, 20 ml of medium was changed by centrifuging the cells at 1200

rpm for 10 min at 4 ºC. The cells were resuspended in to 20 ml of fresh medium and

put back in to culture flask along with 10 ng/ml IL-3. Medium was changed twice a

week till the cells were 4 weeks old. Cells were then checked for mast cell surface

receptors markers, IgE-receptor142 and c-kit143, by flow cytometry.

47


2.2.8 Flow cytometry

Figure 12: Exemplary flow cytometry images of BMcMcs for their characteristic markers c-kit and IgE receptor. Cells were visualized with anti-c-kit-FITC and anti- Fc epsilon receptor I (FcεRI)-PE. Around 34% of cells were

double positive and can be regarded as bone marrow-derived mast cells.

After 4 weeks of culture, mast cells were counted by CASY cell counter. 5 * 105

cells per sample were taken and centrifuged at 2400 rpm for 10 min at 4ºC.

Supernatant was discarded and cell pellet was washed once with MACS buffer at

2400 rpm for 10 min at 4 ºC. Cells were blocked with 1:500 dilution of Fragment

crystallizable of Ig (Fc) block (FcγREC 2.4 g; 5.1 MG/ML, DRFZ) in fluorescence

activated cell sorter (FACS) buffer for 15 min at 4 ºC. After 15 min, cells were

washed by MACS buffer at 2400 rpm for 10 min at 4 ºC. Cells were further stained

by anti-CD117 FITC (c-kit) and anti-FcεRI (IgE) PE antibody 1:50 dilution in 100 μL

cell suspension for 30 min at 4 °C in dark. After 30 min, cells were centrifuged at

2400 rpm for 10 min at 4 °C. Cells were resuspended in to MACS buffer and fixed

by 1 % PFA in PBS for 15 min at 4 °C and then pelleted down at 2400 rpm for 10

min at 4 °C. Cells were resuspended afterwards with 500 µl of MACS buffer and

filtered to remove cell debris. Finally, cells were analyzed by flow cytometry within

24 hr.

48


2.2.9 Stimulation of BMcMCs

For ex vivo and in vitro experiments with mast cell supernatant, 2* 106 BMcMcs

were counted and washed with medium at 2400 rpm for 10 min at 4 °C. Cells were

resuspended and sensitized overnight with 1 µg/ml IgE144. Next day cells were spun

down and resuspended in 2 ml medium and rested for 1 hr. After 1 hr, 1* 106 cells

were seeded per well in a 24 well cell culture plate. One well was stimulated with 1

µg/ml of anti IgE145 for 30 min and other well served as an unstimulated control.

After 30 min, cells were transferred into a 1.5 ml tube and spun down at 2400 rpm

for 10 min at 4 °C. Supernatants were frozen in -80 ° C for further experiments.

2.2.10 Histology and immunohistochemistry

The frozen 5 mm2 of skin biopsies from patch area of mice were cut into 5 μM cross

sections by cryotome at -23 °C to -24 °C. The sections were directly transferred on

microscopic slides and dried on a hot plate for 15 min and stored at -80°C. The formalin treated 5 mm2 of skin biopsies were embedded in to paraffin blocks

and cut into 5 μM cross sections by microtone at room temperature. The sections

were directly transferred on microscopic slides and dried on a hot plate for 15 min.

After 15 min, sides were stored at room temperature.

Figure 13: Example of TSLP and MC staining in the skin of the mice. Cell infiltrates of A) TSLP B) MC respectively. Positive immunohistochemical stained cells were counted at 100X

magnification by using Axiovision software.

49


2.2.10.1 TSLP staining For TSLP staining, 5 mm2 skin sections were deparaffinized by following steps:

Steps Time (min)

Xylol 10

96% EtOH 5

96% EtOH 5

70% EtOH 5

H2O 5

Skin sections were blocked with 1% BSA for 20 min at room temperature and

incubated with H2O2 (Dako, Germany) for 10 min at room temperature to block

endogenous peroxidase activity. Skin sections were then washed 3 times in 1X PBS

with 0.05% Tween 20 (1X PBST) and blocked with the avidin/biotin blocking kit

(Dako, Germany) for 15 min each. Slides were washed as mentioned above and

sections were incubated with goat αm TSLP (clone no.: AF555, R&D Systems) for 1

hr followed by 3 times wash with 1X PBST. Samples were later incubated with

biotinylated SP conjugated affiniPure rabbit anti-goat IgG (Jackson

ImmunoResearch Laboratories) for 30 min at room temperature. Negative controls

were run in parallel omitting either the primary or the secondary antibody. After

washing the slides, sections were developed with the AEC substrate kit (Dako,

Germany) and counter-stained with hematoxylin (Sigma-Aldrich, Germany) to

staining the nuclei as blue and eosinophilic structures in red.

50


Figure 14: TSLP positive cells in the skin lesions upon acute irritation with croton oil for 18 hr.

2.2.10.2 Mast cell staining To stain the mast cells granules containing heparin and histamine (metachromatic),

skin section were rehydrated by 1X TBS for 3 min and stained with 0.1% toluidine

blue in 0.5N hydrochloric acid (HCl) for 1 hr followed by brief washing with tape

water.

Figure 15: Mast cell positive cells in the OVA induced AD skin lesions.

51


2.3 STATISTICAL ANALYSIS

Normally distributed data are depicted as mean ± SEM and non-normally distributed

data are shown as median ± range. Experiments with only two groups were

analyzed using t-test (paired or unpaired) or Wilcoxon matched paired test, when

groups were not normally distributed; for more than 2 groups, depending on the

data distribution, 1-way analysis of variance (ANOVA) was used, followed by

Bonferroni multiple comparisons test or Kruskal-Wallis test. Statistical analyses

were performed with GraphPad Prism version 5 (GraphPad Software, USA). P

value less than 0.05 was considered as statistically significant.

52

Results

3. RESULTS

3.1 SKIN IRRITATION LEADS TO TSLP PRODUCTION

3.1.1 Physical or chemical irritation of the skin leads to production of TSLP in vivo

As many studies have shown that a genetic manipulation of the skin can lead to

elicit TSLP expression in keratinocytes upon skin barrier disruption51,125,128,146, we

speculated whether acute skin irritation would be sufficient to initiate TSLP

production in the skin upon the treatment with physical or chemical irritants in vivo.

For this purpose, mice were subjected to 4 different irritation protocols. A defined

area of the belly and the back were treated for 4 and 18 hrs. The types of irritation

included shaving and tape stripping as mild physical injuries, 1% SDS and croton oil

were used as chemical stimuli. As shown in Fig. 16A, the TSLP gene expression

was induced in all settings, in particular by pure croton oil, which contains phorbol

esters147, followed by wet shaving, SDS and tape stripping. The induction of TSLP

mRNA expression was rapid, as it was mostly induced after 4 hr, but differences

compared to the control were still visible after 18 hr (Fig.16A). This mild irritation on

a reduced skin area was already sufficient to initiate a systemic TSLP protein

response, so that irritated (but not control) mice displayed substantial amount of

TSLP in the serum (Fig. 16B).

Figure 16: Physical and chemical irritation of the skin promotes TSLP production in vivo.

53

Results

A) TSLP mRNA expression in the irritated skin after 4 hr (left panel) and 18 hr (right panel) post-irritation,

respectively. Data are shown as mean ± SEM. B) TSLP protein levels in serum after 4 and 18 hr post-irritation,

each mouse is indicated by a single dot, the bar corresponds to the median+range; n = 7-12 mice/group (*P ≤

0.05, **P ≤ 0.01, ***P ≤ 0.001).

Likewise, TSLP protein levels in skin biopsies were detectable in the irritated skin

by immunohistochemistry (Fig. 17). Overall, even mild skin irritation lead to rapid

production of TSLP protein, which can be detected systemically as it enters to the

circulation quickly, thereby becoming systemically measurable.

Figure 17: Physical and chemical irritation of the skin promotes TSLP production in vivo. TSLP staining by immunohistochemistry in skin section after irritation with tape stripping, shaving, SDS or

croton oil after 18 hrs.

54

Results

3.1.2 Pro-inflammatory cytokines elevate TSLP production in murine KCs

Several factors have been reported to trigger TSLP in human epithelial cells and

skin explants. Such factors include IL-1β, IL-1α, TNF-α, IL-4, IL-25, IL-33 and TLR3

ligand148-151. Little is known about the TSLP drivers in the mouse, an organism

which typically serves to study TSLP function in vivo.

To understand how TSLP may be regulated in murine skin, primary keratinocytes

were isolated from mouse skin and stimulated with several cytokines. PMA served

as positive control. Baseline TSLP expression was found in all experiments. Among

the cytokines studied, TNF-α was able to enhance baseline TSLP production most

efficiently, followed by IL-4 and IL-1β (Fig. 18A). In contrast, IL-33, IL-25, and TLR3

ligand showed little or no effect on TSLP production in murine KCs (Fig. 18A).

Figure 18A: Pro-inflammatory cytokines elevate TSLP production in murine KC. Murine KCs were treated with various stimuli for 24 hr. Cell free supernatant were collected and quantified. Data

are depicted as mean ± SEM of 9-18 experiments. (**P < 0.01, ***P < 0.001).

Pro-inflammatory (TNF-α or IL-1β) and Th2 cytokines have been shown to

synergize regarding TSLP production in human skin explants123 and other epithelial

cells (e.g. human KCs, bronchial epithelial cells, or nasal polyp fibroblasts148,149,152).

Therefore we analyzed whether the same synergism occurs in the current mouse

KC setting. Indeed we were able to detect after incubation with IL-1β + IL-4 and

TNF-α+IL-4 an increased TSLP production (Fig. 18B).

55

Results

Figure 18B: Pro-inflammatory cytokines elevate TSLP production in murine KC. Murine KCs were treated with various combinations of stimuli for 24 hr. Cell free supernatant were collected and

quantified. Data are depicted as mean ± SEM of 9-18 experiments. (*P < 0.05, ***P < 0.001).

The above finding suggests that murine KC differ from human KC in somehow. To

confirm our results we directly compared the data from murine and human KCs. We

found profound differences between the two species regarding TSLP production

(Fig. 18C). As TNF-α failed to induce TSLP expression in human KCs compared to

mouse, while IL-1β was more effective in human KCs; TLR3 ligation was the most

active in human KCs149,153. Taken together, we conclude that overlapping

components of TSLP regulation, but at the same time important differences of the

regulation of TSLP were apparent between murine and human.

Figure 18C: Comparative analysis between murine and human TSLP production.

56

Results

KCs from mouse and human were isolated and stimulated with 10 μg/ml TLR3 ligand, 20 ng/ml IL-1β, 20 ng/ml

TNF-α or 20 ng/ml IL-4. TSLP levels were measured in the cell-free supernatants after 24 hr. The mean ± SEM

from 3-12 experiments is depicted (*P < 0.05, **P < 0.01, ***P < 0.001).

3.1.3 Skin biopsies from mouse and human produce TSLP ex vivo

By using an ex vivo strategy as previously reported for human skin139 and recently

re-established by us for murine skin154, we analyzed next, whether skin biopsies

derived from mice can be stimulated to produce TSLP ex vivo. Upon stimulation,

1% SDS was able to induce a significant amount of TSLP (Fig. 19A), while

scratching induced only a slight TSLP protein expression ex vivo, not reaching

statistical significance. Croton oil was not used for the stimulations due to its

viscous nature and ability to induce a rapid cell death when applied ex vivo (data

not shown). IL-1β, TNF-α and IL-4 induced again TSLP protein as determined in

previous in vitro experiments with mouse KCs. IL-1β was even more a potent TSLP

inducer ex vivo compared to KCs. Similar to the KCs data, IL-33 failed to enhance

TSLP production (Fig. 18A; Fig. 19B).

Figure 19: Chemical irritants and cytokines induce TSLP production ex vivo. Skin biopsies were stimulated for 8 hr with irritants or proinflammatory cytokines ex vivo. TSLP levels were

measured in the supernatants by ELISA, A) after the application of irritants, b) after stimulation with cytokines.

The mean ± SEM from 3-12 experiments is depicted (*P < 0.05, **P < 0.01, ***P < 0.001).

57

Results

3.1.4 IL-1 contributes to SDS-mediated TSLP induction

Irritation experiments revealed that TSLP mRNA and protein production was rapidly

induced upon SDS treatment (Fig. 16A; Fig. 19A). SDS is a known detergent which

can disrupt the skin barrier. This is followed by cell lysis with the release of IL-1α,155-

157 making it a key factor to explain SDS-mediated TSLP induction. To analyze this

hypothesis in more detail we first performed a kinetic study where we found that the

maximal TSLP induction was achieved after 3 hr (data not shown). We used this

system to study the influence of IL-1 by two different approaches. To counteract IL-

1 signalling, in the first approach IL-1Ra was employed158, whereas in the second

approach an IL-1α antibody was used to neutralize IL-1α by a function in SDS

treated skin biopsies in mice. Both approaches resulted in a significant reduction of

SDS-induced TSLP protein production (approximately 20%) (Fig. 20A).

Next, we investigated the influence of endogenous IL-1 on SDS-mediated TSLP

induction in human epidermal sheets considering that IL-1 had much greater effect

on TSLP induction in human compared to mouse KCs (Fig. 18C). By performing a

kinetic study on human epidermal sheets (epidermal sheet was used because total

human skin was too thick for stimulation), SDS elicited TSLP responses in human

epidermal sheets were likewise in murine skin the maximal TSLP induction was

observed after 3 hrs. In human skin, SDS-mediated TSLP induction was reduced

up to ≈50% compared to controls in both settings with the IL-1Ra and the anti-IL-1α

antibody (Fig. 20B).

58

Results

Figure 20: SDS-mediated TSLP induction is IL-1 dependent ex vivo. A) Skin biopsies from mouse were stimulated ex vivo by 1% SDS for 5 min in the presence or absence of

200ng/ml of mIL-1Ra or 25 µg/ml of αmIL-1α-Ab (rabbit IgG served as control). TSLP was quantitated in the

supernatants after 3 hr. The mean ± SEM from 8-10 experiments is depicted (**P < 0.01). B) Epidermal sheets

from human skin were isolated and stimulated ex vivo by 1% SDS for 3 min in the presence or absence of 200

ng/ml hIL-1Ra or 1 µg/ml αhIL-1α-Ab (rabbit IgG served as control). TSLP was quantitated in the supernatants

after 3 hr. The mean ± SEM from 4-5 experiments is depicted (*P < 0.05, **P < 0.01).

3.2 AGGRAVATED AD IN TNF-/- MICE

As the role of TNF-α in AD is not well understood and somewhat controversial, we

investigated this interaction in more detail. AD was induced in TNF-/- mice by

allergen dependent dermatitis. These mice developed a strong dermatitis in

comparison with their wildtype (wt) counterparts, which displayed a mild dermatitis.

The difference between the two groups of mice was highly significant (Fig. 21B).

Conversely, the skin of TNF-/- mice was normal and healthy as wt mice at the

baseline with comparable dermal and epidermal thickness, T cell, MC numbers and

KCs (data not shown, Fig. 24A), suggesting that the development and maintenance

of skin structure does not require TNF-α.

59

Results

Figure 21: TNF-/- mice exhibit increased AD severity. A) Representative illustration of skin lesions with wt and TNF-/- mice AD. C) Quantification of the symptom score

based on different criteria: erythema, extension, dryness, excoriation, crusting (Score 0-3). Each dot represents

single mouse; Median from 3 experiments is depicted (***P < 0.001)

3.3 ROLE OF TSLP IN AD AGGRAVATION UPON TNF DEFICIENCY

3.3.1 Increased TSLP levels in lesional skin of TNF-/- mice and correlation with AD severity

In search of potential AD promoting factors involved under TNF deficiency, we

analysed the local and systemic immune response in these mice. Lower levels of

IgE and IgG1 were observed in the serum of TNF-/- mice. Similarly, neither CD4+

nor CD8+ T cells were changed in lesional skin of the TNF-/- mice (Appendix Fig.

31) compared to wt mice and did not correlate with the symptom score of AD.

Similarly, key Th 1 and 2 cell cytokines, like IL-4, IL-10, IFN-γ and IL-17 were either

equally expressed or slightly enhanced (IFN-γ) in the TNF-/- mice (Appendix Fig.

32). Accordingly, enhanced AD in TNF-/- mice was unlikely due to enhanced T cell

infiltration.

Additionally, other factors like IL-1β (slightly increased), IL-1α, IL-13, IL-33, β-

defensins and skin barrier genes were either comparable or slightly increased in the

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Results

skin of TNF-/- mice and did not correlate with the AD symptom score (Appendix Fig.

33).

Furthermore, we observed a significant increase in of TSLP expression in the TNF-/-

mice (Fig. 22A and 22B). Moreover this finding correlated with the severity of AD as

indicated by linear regression analysis (Fig. 22C). Since TSLP is an important

regulator of AD manifestation, this result implies that TNF can counter regulate AD

development by diminishing TSLP production in vivo, which supports the concept of

TSLP as an excellent candidate to explain AD aggravation under TNF deficiency.

Figure 22: Significant increase of TSLP in TNF-/- mice and its correlation to severity. Allergen triggered dermatitis was induced in to wt and TNF-/- mice. On day 71, mice were sacrificed and lesional

skin was further analysed for A) TSLP mRNA level B) TSLP protein level C) correlation of TSLP mRNA with the

clinical severity of the dermatitis. Median of n = 14 mice/group. (***P < 0.001).

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3.3.2 Anti-TSLP protect TNF-/- regarding AD onset

To further investigate, whether TSLP is responsible for AD development under TNF

deficiency, we used neutralizing antibodies in the AD model to interfere with TSLP

function. Therefore mice were injected with anti-TSLP antibodies on days 41, 45, 62

and 66 (Fig. 9). Upon anti-TSLP application, the onset of AD was diminished,

compared to the wt controls. TNF-/- treatment with appropriate isotype controls

resulted in the development of a strong dermatitis as observed earlier (Fig. 23A).

The clinical score of the AD was significantly reduced in TNF-/- mice when treated

with anti-TSLP compared to its isotype control (Fig. 23B). The TEWL measurement

revealed similar results (Fig. 23C). TNF-/- mice treated with anti TSLP still displayed

TSLP mRNA in lesional skin, (Appendix Fig. 34A) which was not unexpected.

These data clearly indicate that anti TSLP treatment results in an altered TSLP

function but not its expression. Moreover, TNF-/- mice treated with anti TSLP

showed a decrease of MCs numbers in lesional skin compared to an isotype control

group (Appendix Fig. 34B).

Figure 23: Protection of AD in TNF-/- mice upon anti TSLP treatment. A) AD suppressed in TNF-/- mice by intradermal doses of anti-TSLP antibodies B) symptom score C) TEWL

which represents a characteristic of dermatitis. Median of n = 6-9 mice/group. (**P < 0.01).

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Results

3.4. ENDOGENOUS TNF-α DOES NOT CONTRIBUTE TO TSLP PRODUCTION

TNF is a potent inducer of TSLP in vitro, as shown by our data from murine skin

(Fig. 18A-C, Fig. 19B). As the role of TNF-α is poorly defined in vivo and as it is not

clear if TNF-/- mice are inherently prone to produce increased levels of TSLP, we

studied next the impact of endogenous TNF-α on TSLP expression by analysing

TNF-/- compared to wt mice.

As mentioned above, we confirmed that the untreated skin of TNF-/- mice was

comparable to their wt counterparts (data not shown). Purified KCs from TNF-/- and

wt were not morphologically distinguishable (Fig. 24A). KCs from both strains show

a comparable growth and survival rate (data not shown). This suggests that TNF-α

does not have a direct influence on KC differentiation.

Surprisingly the in vitro data shows that a comparable amount of TSLP was

produced by TNF-/- KCs compared to wt KCs upon stimulation with IL-1β (Fig. 24B),

exogenous TNF-α and IL-4 (data not shown).

Similar results were obtained ex vivo; skin biopsies from both strains show

comparable levels of TSLP production when treated with physico-chemical irritants

(Fig. 24C) and proinflammatory cytokines (Fig. 24D) replicating the in vitro results.

Finally, to verify that endogenous TNF-α was not required for increased TSLP

production in mice, we performed an in vivo study with acute skin irritation. This

data reveal that TSLP production was unaffected under TNF deficiency at the

mRNA (Fig.24E) and protein levels (Fig. 24F).

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Results

Figure 24: Endogenous TNF is not required for TSLP production in murine skin. In vitro: Primary KCs were isolated from wt and TNF-/- mice and compared for TSLP levels; A) Representative

illustration of primary KCs from wt and TNF-/- mice. B) TSLP levels in cell free supernatant stimulated with

20ng/ml IL-1β after 24 hr. Ex vivo: Comparison of skin biopsies from wt and TNF-/- mice regarding TSLP

production induced by C) physical or chemical irritation and D) cytokine mediators after 8 hr. In vivo: wt and

TNF-/- mice were subjected to different irritants E) TSLP mRNA levels in skin. F) TSLP protein levels in serum

after 18 hr. Median of n = 8 mice/group. Mean ± SEM is depicted from 4-9 experiments.

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Results

3.5 MAST CELLS CONTRIBUTE TO TSLP PRODUCTION

3.5.1 MCs are increased in lesional skin of TNF-/- mice and correlate with AD and TSLP

Our data suggest that increased levels of TSLP under TNF deficiency were not the

result of an inherently prone TNF-/- mice to over express TSLP, but may requires an

inflammatory micromilieu as present in AD. As MCs play an important role in AD, we

performed an analysis of MC in lesional skin. Indeed, MCs numbers were

significantly increased in TNF-/- mice compared to wt mice (Fig. 25).

Figure 25: MCs are increased in lesional skin of TNF-/- mice A) Representative illustration of MCs numbers in the lesional skin of TNF-deficient mice. B) MCs numbers in the

lesional skin of TNF-deficient mice as compared to wt. Median of n ≤ 14 mice/group. (***P < 0.001).

Moreover, we determined a significant correlation of the severity of dermatitis (Fig

26A) as well as the TSLP expression level in lesional skin (Fig. 26B) by linear

regression analysis.

Stem cell factor (SCF) and its receptor c-kit are well known markers for the growth

and survival of MCs 159. Therefore, we measured the mRNA expression of SCF and

c-kit in lesional skin. SCF was slightly increased in the TNF-/- mice compared to wt

and c-kit expression remained unchanged (data not shown).

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Results

Figure 26: MC numbers in the lesional skin correlates with the severity of AD and with TSLP mRNA expression. Correlation of MCs with A) symptom score and B) TSLP mRNA expression respectively. Median of n ≤ 14

mice/group. (***P < 0.001).

3.5.2 Anti c-Kit is protective for AD development in TNF-/- mice

Since, MCs numbers were increased in the AD model and correlated with the skin

score as well as with TSLP expression, further experiments were performed to

understand the role of MCs in the AD model by application of c-kit neutralizing

antibodies to interfere with MC increase. TNF-/- mice treated with anti c-kit

antibodies show a milder appearance of AD under TNF deficiency (Fig. 27A).

Accordingly, the skin score was reduced in TNF-/- mice, when treated with anti-c-Kit

antibody compared to its isotype control, though this data did not reach statistical

significance (Fig. 27B). The data from TEWL measurements indicated similar

results (Fig. 27C).

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Results

Figure 27: Anti c-Kit antibody treatment alleviates AD symptoms in TNF-/- mice. A) AD model depicting treatment with anti c-Kit ab. B) AD slightly suppressed in TNF-/- mice by intradermal

administration of anti-c-Kit ab C) AD skin score D) TEWL, which represents a characteristic of dermatitis.

Median of n = 5-12mice/group. (**P < 0.01).

3.5.3 MCs do not produce a relevant amount of TSLP

We investigated next how MCs contribute to TSLP production. Published studies

suggest that mast cells can produce and respond to TSLP160. To confirm MCs as a

source of TSLP under TNF deficiency, BMcMCs were sensitized with IgE and then

stimulated with anti IgE in the presence or absence of either IL-1β or TNF-α or with

their combinations (Fig. 28A). TSLP was measured at the protein level. The data

clearly indicate that BMcMCs are not able to produce TSLP upon stimulation (Fig.

28B) either when driven from wt or TNF-/- mice. This data confirm that MCs are

unlikely to contribute significantly to an increased TSLP under TNF deficiency in an

inflammatory micromilieu.

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Results

Figure 28: MCs are not producing relevant amounts of TSLP. A) BMcMCs from wt and TNF-/- mouse were sensitized overnight with IgE and stimulated of anti IgE in the

presence or absence of either IL-1β or TNF-α or with their combinations for 24 hr. B) TSLP protein levels in cell

free supernatant. The mean ± SEM from 3 experiments is depicted.

3.5.4 MCs as instructors of TSLP production by KCs

Based on literature and our data, it is clear that MCs are not potent producers of

TSLP133. We hypothesized that MCs may be able to instruct KCs to produce TSLP

under TNF deficiency as KCs are the most potent TSLP producers. This hypothesis

might also provide a substantial link between increased TSLP levels and MC

numbers which we observed in murine AD skin lesions from TNF-/- mice.

3.5.4.1 Resting MCs supernatant enhanced TSLP levels ex vivo

To validate our hypothesis, we performed an ex vivo experiment where we

stimulated skin biopsies with supernatants from stimulated or unstimulated (resting

MCs) BMcMCs with anti IgE for 30 min. Supernatants from stimulated BMcMCs

were not able to enhance the TSLP levels significantly, moreover higher amounts of

the supernatants (2%) resulted even in a slight inhibition of TSLP production (Fig.

29A). On the other hand, supernatants (1%) from unstimulated MCs promoted a

significant increase of TSLP production in murine skin biopsies (Fig. 29B), indicating

that MCs can instruct KCs to produce TSLP.

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Results

Figure 29: Supernatants of resting MCs instruct KCs to produce TSLP. The stimulated supernatant of bone marrow-derived mast cells, stimulated with anti-IgE or unstimulated

(resting) for 30 min, was incubated with skin biopsies for 16 hr. TSLP protein levels with A) 30 min stimulated

MCs B) unstimulated MCs (resting MCs). The median from 5-8 experiments is depicted (**P < 0.01).

3.5.4.2 mMCP6 significantly increased TSLP protein levels in skin ex vivo

The results show that supernatants from resting mast cells resulted in significantly

increased TSLP protein expression. The next question was which MC factor might

be responsible for this finding. From the literature it is known that MCs can release

many different mediators upon degranulation161. The most predominant MCs

mediators are histamine and β-tryptase, which are equivalent to the mouse mast

cell protease 6 (mMCP6). Therefore we analyzed next the impact of histamine and

mMCP6 on TSLP expression.

Skin biopsies stimulated with histamine did not affect TSLP expression (data not

shown), while mMCP6 significantly enhanced the TSLP production starting at 10

ng/ml (Fig. 30). A higher concentration of mMCP6 led to inhibition of TSLP

production as observed earlier when the stimulated MCs supernatants were used.

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Results

Figure 30: Mouse mast cell protease 6 (mMCP6) promote skin derived TSLP production ex

vivo. A) Stimulation of skin biopsies with mMCP6 for 16 hr, significant increase of TSLP protein levels stimulated with

concentration of 10 ng/ml. The median from 5-8 experiments is depicted (**P < 0.01).

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Discussion

4. DISCUSSION

TNF-α is a well-known pro-inflammatory cytokine which plays a crucial role in

inflammatory diseases80. The role of endogenous TNF-α in skin inflammation and

particularly AD is not well understood. Based on the literature, an adverse

interaction between TNF and AD was described and suggests that in the absence

of TNF, AD seem to be enhanced87-89. On the other hand, TSLP which is directly

produced by keratinocytes is considered to be the initiator of the disease.

Previously, it has been shown that TSLP overexpression in mouse skin promotes

the development of a spontaneous dermatitis resembling characteristics of human

AD49,103. The role of TSLP in the development of allergic disease is well understood,

however the influence of endogenous TNF-α on TSLP production or its activation is

not yet clear.

In this thesis the irritation-induced TSLP production and its role in AD progression

was investigated. We also tried to better understand the role of endogenous TNF-α

in relation to TSLP production under irritative stimuli but also in an environment

which is present in AD. Another important component of AD pathogenesis are MCs.

They have been shown to be increased in lesional skin from AD patients but also in

lesional skin from AD mice162. To explore the role of mast cells as a trigger for TSLP

production by keratinocytes a mast cell depleting antibody was used in vivo and

mast cell supernatants but also mast cell mediators were analyzed in more detail ex

vivo.

4.1 SKIN IRRITATION LEADS TO RAPID INDUCTION OF TSLP, INDEPENDENT FROM TNF-α, BUT PARTIALLY DEPENDS ON IL-1

A disrupted barrier makes the skin more susceptible to the environment as shown

by Mogbekeloluwa O Danso et al. (2014)163. This allows allergens or irritants to

enter through the skin and to induce a Th2 response which in turn activates

keratinocytes to produce TSLP to activate dDCs thereby promoting the

inflammatory process. Many genetically modified mouse models have been used to

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Discussion

show an essential role for TSLP in allergic diseases60,129. As our understanding

about functional aspects of TSLP in different pathophysiological conditions is slowly

increasing, the regulatory role of endogenous TSLP has been hardly well-defined.

Moreover, there are a lot of discrepancies between in vitro and in vivo studies164.

Various genetic studies with transgenic mice show that gene manipulation of

different genes in the skin was followed by an increase of the TSLP

expression51,125,146,165-167. The manipulation of different unrelated genes such as

Notch and lymphoepithelial Kazal-type-related inhibitor (LEKTI) display similar

outcomes. Therefore we hypothesized that skin perturbation caused by either

genetic manipulation or by environmental factors can initiate a specific cascade that

leads to TSLP production, even outside of an allergic scenario.

To further investigate our hypothesis and to understand whether barrier disruption

would be sufficient for the initiation of TSLP production in mouse skin, a range of

irritants were used. The data show that TSLP induction was an elicited as a

common consequence in inflamed skin when skin homeostasis was deviated. This

observation was made and confirmed by the variety of TSLP inducers, ranging from

physical trauma i.e. taking punch biopsy of the skin to mild physical irritation (wet

shaving, tape stripping) and chemical insults (croton oil, SDS). These data was

supported by a previous finding with the detergent SDS. SDS is used in different

models as irritants and can alter the stratum corneum due to its action on surface

tension. Therefore it has been used in different patch tests models and animal

assays as it is able to enhance the penetration of other substances168. Moreover

SDS can cause to a large extend alterations of the skin barrier function169. On the

other hand, croton oil has been used to induce ear edema in a mouse model170,171.

One important component of croton oil is the phenol ester 12-O-

tetradecanoylphorbol-13-acetate (TPA). Furstenberger et al. (1994) have shown

that topical application of croton oil can initiate local inflammation accompanied by

edema formation, polymorphonuclear leukocyte infiltration and epidermal

hyperproliferation, as a consequence of the production of inflammatory mediators,

such as prostaglandin E2, leukotrienes, histamine, serotonin and IL-1172. The non-

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Discussion

specific skin inflammation elicited by SDS and croton oil resemble early phase

events during AD development in certain mouse models173,174.

In accordance with the literature, our data show a link between TSLP expression in

skin even in the absence of any particular allergic scenario. 2010, Angelova-Fischer

et al. show that injury of the stratum corneum by tape stripping and 2% SLS leads to

an increase of TSLP in human epidermis117. 2007, Allakhverdi et al. observed that a

trauma driven by a punch biopsy was sufficient to induce TSLP expression in

human skin122. These findings are in line with our data, derived from the acute

mouse skin model. Using this model we show that the increase of TSLP mRNA in

the skin but also protein in the skin and the serum was more pronounced after

physical irritation although the highest increase of TSLP expression was determined

after croton oil application. These data were further confirmed in our ex vivo cell

culture settings. Additional, evidence for the susceptibility of the TSLP gene to

various insults are known from physical irritation, UV irradiation, malignancy,

xerosis, and even in the absence of intestinal microbiota117,175-180. In support to

previous observations, our acute skin mouse model not only revealed that the

different types of stressors can lead to TSLP induction, but we could also for the

first time that TSLP induced after mild skin irritation can be measured in the serum

within in a short period of time i.e only after 4 hrs and increases over the time (18

hrs).

In search of a mechanism by which the gentle irritation and other distress can

induce TSLP in murine skin, we first looked for factors which can induce TSLP in

murine skin. As mentioned above several factors have been described to induce

TSLP in human keratinocytes. These include e.g. TNF-α, IL-1, IL-4, IL-25, IL-33 and

TLR3-ligand123,148,149,151, although their exact effects in murine keratinocytes are not

well known in detail yet. Our in vitro and ex vivo findings indicate that primary

keratinocytes and skin explants are capable to produce TSLP upon treatment with

different stimuli in a similar manner as in humans in spite of some unexpected

results. These findings are in accordance with a report from Takai et al. (2012)164.

These authors demonstrate that the gene expression of TSLP was induced in

bronchial epithelial cells (NHBE) after the exposure with different proinflammatory 73

Discussion

cytokines such as IL-1β, TNF-α and TLR2, TLR8, and TLR9 ligands. They also

indicated that the effects of these cytokines in both species are derived from the

NFκB pathway. In contrast, our data from the skin epithelial cells show that there is

considerable species dependence. TNF-α for example was able to induce TSLP to

a greater extend in mouse keratinocytes compared to human keratinocytes. By

contrast, IL-1β was more active in human keratinocytes compared to mouse. In

addition, TLR3 ligand was significantly stronger effective in human than in mouse

keratinocytes as described previously149,153.

In vitro studies with human primary keratinocytes show that cytokines like TNF-α in

combination with Th2 cytokines such as IL-4 and IL-13 can synergistically increase

the poly I:C (TLR-3 ligand) induced TSLP149. 2007, Bogiatzi et al. also found similar

results from human skin explants stimulated with different combinations of pro-

inflammatory cytokines such as TNF-α or IL-1α, in combination with Th2 cytokines

such as IL-4 and IL-13123. These results were further confirmed by many other

groups using different cell types. For example, Allakhverdi et al. (2007) show in

human airway epithelial cells, that a combination of TNF-α and IL-1α augmented

TSLP expression122. In accordance with these findings, we were able to confirm this

data in our in vitro and ex vivo settings. TSLP expression was increased in mouse

keratinocytes and in skin explants upon treatment with TNF-α and IL-1β in

combination with IL-4.

Finally, we addressed the mechanisms by which the acute skin irritation and other

physical stress can elicit TSLP in murine skin. TNF-α was the primary candidate to

be investigated as this cytokine is well-known to be induced upon skin irritation82,156

and to increase TSLP in the skin123. This was also observed in our in vitro and ex

vivo results making TNF-α to a probable intermediary in the cascade to mount

TSLP production. However, surprisingly we found that endogenous TNF-α was

expendable for TSLP production under all different settings i.e in vitro, in vivo and

ex vivo.

Taken together, it is obvious that exogenous TNF-α can induce TSLP in murine skin

under different settings, but endogenously it is not able to reach threshold levels

which may be required for its effect during skin damage. Although the impact of 74

Discussion

TNF-α regarding TSLP production in human compared to mouse skin was lower in

vitro, we investigated the role of endogenous TNF-α upon skin perturbation in

human epidermal sheets. As expected, endogenous TNF-α induced lesser TSLP

protein in the human in comparison to the mouse system emphasizing on its

dispensability in both the murine and the human skin.

Since TNF-α was found to be not required for TSLP production in the skin, an

alternative mechanism was needed to be identified. Upon trauma cells may die and

defined defense and repair processes in the respective host are activated. Such

processes or pathways include in part the activation of the ancient IL-1 family181,182.

Previous studies have shown that cytokines from the IL-1 family are involved in

SDS-mediated skin irritation156. Physical disruption of the skin can induce the

release of IL-1α. The dispensability of TNF-α and based on the literature, IL-1

seemed to be a reasonable candidate to explain the present findings. By using IL-

1Ra and IL-1α neutralizing antibodies, IL-1 was certainly shown to participate in

TSLP production in the skin when triggered by SDS. Around 20% of TSLP

production was reduced when treated with IL-1Ra or by the according neutralizing

antibodies. This indicates that IL-1 is involved in SDS mediated TSLP production in

damaged skin. As the reduction of TSLP reached only 20% we believe that other

factors may be also be involved and are likely to play an additional role in the TSLP

production upon SDS treatment. However, the other IL-1 family member IL-33183 is

not likely to be a suitable candidate as it was not able to induce TSLP in skin

constituents. Other new members of the IL-1 family whose specific contribution to

inflammatory skin conditions has only begun to be cleared182. So it is still not clear

whether these mediators such as high mobility group box chromosomal Protein 1

(HMGB1)184 might serve as a driving force for TSLP production in skin.

Nevertheless, IL-1 had a better impact on the irritation-induced TSLP production in

human skin, which was in the range of around 50%. The higher responsiveness of

human KCs towards IL-1 compared to murine KCs suggest that IL-1 is more

important in human skin compared to mouse skin. Therefore TSLP regulation in

different species will not be fully the same185. The apparent species variation in the

actions of TSLP provides another example of significant differences between the

human and murine immune system185. This is further supported by our observation 75

Discussion

that murine KCs were basically resistant to TLR3-mediated TSLP induction

compared to human KCs, although murine skin was susceptible to TLR3-ligands in

other settings186.

In summary it can be proposed based on the findings from this thesis that TSLP

acts as an executer of the innate alarm system aiming to protect the host defense

and restoring barrier function. That TSLP is critical to healing and barrier restoration

in mucosal tissues187-189, providing anti-tumor and antimicrobial effects in the skin

has already been demonstrated175,176,190 and that is moreover able to induce

extramedullary hematopoiesis191. Therefore it seems that the role of TSLP outside

of the allergic scenario will require a thorough investigation in the future.

4.2 TNF-/- MICE DEVELOP AGGRAVATED AD AND DISPLAY INCREASED TSLP EXPRESSION AND MCs NUMBERS CORRELATING WITH DISEASE SEVERITY

AD is a chronic inflammatory skin disease with a complex pathogenesis. It is

caused by a combination of an epidermal barrier dysfunction and an immune

dysregulation163. A variety of factors contribute to the pathogenesis of AD, including

environmental or genetic factors. Immunological factors as well as pharmacological

abnormalities play a role in disease progression192.

TNF-α is known for its pleiotropic functions in host defense but also for its role on

the elicitation of inflammatory diseases during immune dysregulation79,80. Anti-TNF

therapies are approved and effective for autoimmune disorders such as rheumatoid

arthritis, inflammatory bowel disease and psoriasis with relatively few side-effects77.

The two monoclonal antibodies adalimumab and infliximab and the soluble receptor

etanercept are the most commonly prescribed therapeutics in autoimmune diseases

interfering with the TNF function193,194. A direct role of TNF-α in AD initiation and

development is not well understood. A detailed analysis of the literature revealed

more evidence for negative than a positive association. 1992, Takahashi et al, has

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Discussion

shown that the PBMCs from AD patients had decreased levels of TNF-α compared

to healthy controls86. Nomura et al. (2003) also observed lower levels of TNF-α, IL-

1β and IFN-γ in the skin from AD patients when compared to skin samples derived

from psoriasis89. Similarly dermal inflammatory DCs which produce TNF-α were

found to be reduced in the skin of AD vs psoriasis patients195. The most striking

evidence came from epidemiological studies from different clinics including our

institution, which reported the onset of AD like symptoms as a side effect of anti

TNF therapy90,91,196-198. These findings point towards a rather protective role of TNF

in the context of AD, but the experimental evidence was missing. To support

previous findings and clinical observations we used a murine model. To delineate

the underlying mechanisms, we applied an OVA-allergen AD mouse model in TNF-/-

mice to mimic the human AD scenario. Indeed, TNF-α protected against AD

development as reported in literature as worsening of the severity of AD-like

lesions occurred in TNF-/- mice. The measurement of the severity in the AD model

was adopted from the SCORAD score a tool to assess clinical severity in human

AD199. The majority of AD in human is characterized by increased levels of IgE

(extrinsic AD) and the presence of mostly Th2 cells in lesional skin32. Woodward,

A.L et al (2001) have shown that T cells play a critical role in skin inflammation200.

The key role of effector T cells and their major relevance in AD pathology was

further supported by the fact that under T cell immunodeficiency, elevated IgE levels

with eczematous skin lesions were observed32. Other studies have shown that

epidermal CD8+ cells are involved in the pathogenesis of AD201. In search of

mechanism behind AD their accumulation under TNF absence and also to

understand the clinical observations, various factors were analyzed. The analysis of

the humoral immune response from sera of mice from the AD model revealed a

decrease of the specific IgE and IgG1 concentrations in the TNF-/- mice whereas no

change was observed in infiltrating CD4+ and CD8+ T cells.

AD patients have shown to have higher frequencies of Th1, Th2, Treg and Th22

subsets as reported by Turner et al. 2012202. In addition these cells show a higher

expression of IL-5, IL-13, IL-1β IL-4, IFN-γ, IL-12, Gm-CSF, IL-10, TGF-β and IL-

688. The analysis of different proinflammatory cytokines and Th subsets; Th1, Th2,

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Discussion

Treg, Th17 (e.g. IL-4, IL-10, IL-17, IFN-γ IL-1β, IL-1α, IL-13, IL-33 and β-defensins)

revealed no impact or slight increase on their expression under TNF deficiency

indicating that T cells were most likely not responsible for AD aggravation under

TNF deficiency.

Barrier dysfunctioning is well linked to AD pathogenesis. Aioi, A. et al. (2001) have

shown a crucial role of skin barrier function in AD manifestation using NC/Nga mice,

a spontaneous mouse AD model displaying skin barrier abnormalities like increased

TEWL and an abnormal skin conductivity under conventional conditions, but not

under specific pathogen-free conditions203. Similarly, Gupta et al. (2007) have

shown that the pathogenesis of AD is at least in part related to barrier

dysfunction204. In line with these findings, we investigated the expression of several

skin barrier genes in these mice. The expression level of transglutamase, involucrin,

loricrin and filaggrin were either comparable or slightly increased in TNF-/- mice and

did not correlate with AD symptom score (data not shown).

TSLP is a well-known key initiator of allergic diseases including AD and asthma126.

TSLP has been shown to be increased in lesional skin from AD patients but not in

nickel induced contact dermatitis103. TSLP is an IL-7 type of cytokine belonging to

the IL-2 family, which can act on DCs and can promote Th2 cell differentiation and

recruitment. TSLP can also directly act on naïve CD4+ cells to promote proliferation

in response to antigen114. Ziegler et al. (2013) demonstrated that epithelial cell-

derived TSLP can activate T cells, DCs and mast cells205. The overexpression of

TSLP in murine skin leads to the development of a spontaneous dermatitis49,103. In

agreement with the literature, TSLP was significantly overexpressed in AD skin and

also correlated with disease severity. TNF-α is known to regulate TSLP expression.

2011, Brandt and Sivaprasad provided evidence that human skin explants produce

higher levels of TSLP, when treated with a combination of pro-inflammatory

cytokines i.e. TNF-α, IL-1α and Th2 cytokines i.e IL-4 and IL-13, but not alone44.

Similar results were obtained from keratinocyte cultures149,206.

To understand why TSLP was enhanced in TNF-/- mice although TNF-α is a well-

recognized inducer of TSLP164 different factors and cell types were evaluated in 78

Discussion

TNF-/- mice. Another presumably important component in AD pathogenesis is the

mast cell. Various studies have shown that mast cells are commonly increased in

human but also the AD mouse model162. Therefore we analyzed the mast cell

numbers in lesional skin from the TNF-/- mice.

Mast cells, but not T cells as mentioned earlier were increased in AD lesions of

TNF-/- mice. Its number not only correlated with disease severity but also with

mRNA levels of lesional TSLP in the skin. In agreement with this finding Yong-Jun

Liu et al. (2006) also described that mast cells activated by IgE receptor cross-

linking express high levels of TSLP, which may support allergic inflammation121.

Interestingly, Na-Ra Han et al. in 2014 have shown as well that numbers of mast

cells in different organs of wildtype mice were significantly higher compared with

TSLP-deficient mice as well as TSLP was capable of inducing the proliferation and

differentiation of mast cells207.

4.3 ENHANCED TSLP LEADS TO AD MANIFESTATION

Th2 cytokines, IgE, mast cells, eosinophils and an increased expression of TSLP

are the common characteristics of AD pathogenesis127. Genetic screening of atopic

populations has shown an association between TSLP gene polymorphisms and AD,

clearly suggesting that TSLP plays an important role in atopic diseases208. Since

our AD model show an exaggerated TSLP expression under TNF deficiency, we

wondered if TSLP was responsible for AD development in TNF-/- mice. Saenz SA.

et al. (2008) reported that TNF-α is well-known as a positive regulator of TSLP in

vitro151. These findings are somewhat contradictory with our data were we found a

significant increase of TSLP levels under TNF deficiency. The administration of anti-

TSLP into the skin shows a clear-cut improvement of the symptom score in TNF-/-

mice, compared to the mice which were treated with its isotype control. This finding

is clearly indicating that TSLP plays a key role in the development of AD under TNF

absence. Another critical hallmark of barrier dysfunction is TEWL. An improvement

of the symptom score in AD skin is associated with a decrease in TEWL204. The

measurement of TEWL to backup the data from the symptom score show indeed a 79

Discussion

decrease of TEWL upon treatment with neutralizing TSLP antibodies which fail to

do so in the presence of an isotype control. In agreement with our data,

He and Geha (2010) have demonstrated that TSLPR-/- mice fail to develop allergic

skin inflammation in an AD mouse model upon repeated EC sensitization with OVA

after tape-stripping. They also show the blockade of TSLP by a neutralizing

antibody that inhibits the development of allergic skin inflammation, suggesting that

TSLP can amplify allergic skin inflammation during the effector phase by acting

directly on skin infiltrating T cells to induce Th2 cytokine secretion60.

The expression level of TSLP in TNF-/- mice after neutralization with anti TSLP

antibody was similar to the TSLP expression as observed after application of the

isotype control. These results indicate that the functional role of TSLP has been

altered with neutralizing antibody instead of inhibiting its production by the cells.

Ziegler and Artis (2010) hypothesize that TSLP can activate mast cells to further

produce increased levels of different cytokines such as IL-13127. In accordance with

these findings, we observed that the knockout mice treated with anti TSLP

displayed a decrease in MCs numbers in lesional skin compared to the isotype

control. This was most likely a result of an altered functional activity of TSLP after

the application of anti-TSLP.

Next the question arose, whether TNF-/- mice are prone to induce increased levels

of TSLP in general (e.g. upon trauma and skin irritation) or whether they require a

specific micromilieu as present in the AD skin. As pointed out earlier endogenous

TNF-α was not required for TSLP production, and its absence did not promote

TSLP over-expression upon acute skin irritation. These findings clearly indicate that

the milieu of AD was most likely necessary for the more pronounced production of

TSLP in lesional skin of TNF-/- mice.

80

Discussion

4.4 MCs SEEM TO PLAY A ROLE BETWEEN TNF-DEFICIENCY AND TSLP

Based on previous findings we hypothesize that TSLP is the trigger of AD under

TNF deficiency through an indirect micromilieiu mechanism as TNF can directly

induce TSLP as described earlier209. The increase of the number of mast cells in

lesional skin of TNF-/- mice and its significant correlation with the severity clearly

indicate that MCs are a hot candidate to explain the relationship between TNF-

deficiency and TSLP. Based on previous literature we assumed that MCs are

upstream of TSLP and downstream of TNF in this scenario. MCs have been linked

with TSLP in different contexts such as they are known to produce TSLP

themselves103,210, are responsive to TSLP122, and can enhance TSLP production by

epithelial cells133. Based on this literature, we aimed to clarify whether an

experimental manipulation of MC density or abrogation of MC function is facilitating

amelioration of AD.

Intradermal administration of cKit neutralizing antibodies to TNF-/- mice, resulted in

an improvement of the symptom score of the TNF-/- mice, compared to their isotype

control. Similar results were obtained with measurements by TEWL although these

data did not reach statistical significance due to a high variation within the groups.

The decrease in the symptom score of AD as well as in TEWL observed in mice

treated with cKit antibodies indicate that mast cells are involved in the process of

AD aggravation under TNF deficiency. Recently, Tomoaki Ando et al (2014) also

have demonstrated that mast cells but not the B or T cells are crucial for the onset

of spontaneous dermatitis in Plcb3-/- mice211.

Our next question was whether mast cells might contribute to TSLP production.

MCs are known to produce TSLP themselves103,210,212. Additional data from Yong-

Jun Liu (2006) suggest that mast cells which were activated by IgE receptor cross-

linking express high levels of TSLP121. In accordance to the above findings, we

activated BMcMcs with IgE cross linking in combination with other cytokines.

BMcMcs from TNF-/- mouse as well as wt mice failed to produce TSLP, indicating

that under TNF-/- deficiency MCs are not likely to be the major source of an

81

Discussion

increased TSLP production in an AD environment. Thus, we speculated that as

MCs are not able to produce TSLP, but rather may act as inducers of TSLP

production since they are also known to regulate epithelial cells regarding TSLP

expression in allergic rhinitis133.

Next our aim was to investigate the role of mast cells as triggers of TSLP production

by instructing keratinocytes as they are the best known TSLP producers127. For this

purpose mast cell supernatants and mast cell mediators were studied regarding the

onset of TSLP production. Different experimental settings with skin biopsies and

MCs were used to pin down the molecular cascade which promotes TSLP

production and consecutively support AD aggravation in a TNF deficient

environment.

Stimulation with anti-IgE led to crosslinking of the FcεRI and hence MCs

degranulate which progressively leads to the release of MC mediators like

histamine and proteases213,214. To investigate the role of MCs mediators that may

enhance TSLP expression by acting on keratinocytes in the skin, skin biopsies were

stimulated with the supernatants of either IgE cross linked stimulated or resting MCs

ex vivo. Stimulated mast cells were not able to enhance TSLP levels. This

implicates that high concentrations of MC mediators in the supernatants may even

inhibit TSLP production compared to their non-stimulated counterparts. This was

also observed by Okayama et al., 2009. These authors have shown that TSLP can

be degraded by MC-derived proteases210. Moreover, MCs release a plethora of

other mediators upon stimulation215-218 which can all have an impact on TSLP

production in the skin. Based on that, it is clear that mast cell supernatants contain

highly concentrated MC-derived proteases which can further promote TSLP

degradation.

Nevertheless, the supernatants from resting MCs contain only mediators released

spontaneously (e.g. through so called piecemeal degranulation219 (Dvorak and

Kissell, 1991)), granula-associated mediators are at lower concentrations in these

supernatants, and they are also virtually devoid of activation-induced mediators like

LTC4 and PGD2220. Interestingly, the supernatants from resting mast cells 82

Discussion

significantly increased TSLP levels in skin biopsies. These data allow to presume

that some of the MCs mediators instruct KCs to produce enhanced TSLP under

TNF deficiency.

In order to identify which factor of the mast cell supernatant may be responsible for

the increased TSLP expression by keratinocytes, the two most abundant mast cell

mediators, histamine and tryptase, were evaluated for their ability to enhance TSLP

expression.

Histamine failed to increase TSLP expression at protein levels. A dose response of

histamine was performed, but no single concentration elicited significant TSLP

expression. Histamine is believed to be a major player in the crosstalk between

mast cells and keratinocytes221,222. For example, human keratinocytes and

organotypic skin models revealed that histamine is able to down regulate the

expression of differentiation-associated proteins like filaggrin, keratin and loricrin, as

well as tight junction and desmosomal junction proteins223. These data suggest that

mast cell activation and histamine release contribute to skin barrier defects which

have been implicated in the initiation of AD223. In addition, histamine is one of the

major pruritogenic factors. Itch is a hallmark of AD, whereas histamine 1 receptor

(H1R) antagonist cannot ameliorate the itch in lesional skin of AD patients224. In

contrast it has been shown in a model of chronic allergic dermatitis in NC/Nga mice,

that the combined treatment of a H1R and histamine 4 receptor (H4R) antagonist

displayed anti-pruritogenic and anti-inflammatory effects225. Based on this evidence,

it is clear that, one of the most abundant mast cell mediators does not appear to

play a role for increased TSLP levels in AD.

The next major factor is the protease β-tryptase which is expressed in mast cells226.

Mast cell proteases constitute between 30-50% of the total mast cell protein

content218. Therefore this mast cell mediator was analyzed next as a possible

trigger of TSLP. The mouse analogue of human β-tryptase is mMCP6, which was

indeed resulting in an increased TSLP production.

83

Discussion

Based on our data it is difficult to answer the question whether tryptase is the main

component from the MC supernatant that triggers TSLP in skin. The application of

anti mMCP6 neutralizing antibody needs to be used to neutralize the induction of

TSLP by either resting mast cell supernatants or by mMCP6. Mast cells from

mMCP6-/- mice would be also required to confirm these data. But in support,

Thakurdas et al. (2007), showed that mMCP6 is not essential for migration,

retention and overall maturation of MC-committed progenitors in connective tissues

in mMCP6-/- mice227. These mice however had a reduced ability to combat K.

pneumonia infections, suggesting a critical immune protective role of mMCP6 in

bacterial infections227. Interestingly, it was reported that tryptase-like enzymes in the

stratum corneum were highly unregulated in lesional AD skin228. In addition, trends

of elevated tryptase levels in MCs from AD patients were observed229. Although,

mast cell tryptase serum levels were investigated for their suitability as a serum

marker for AD, two studies have shown no correlation between mast cell tryptase

serum levels and the severity of AD230,231. However, tryptase has been more and

more implicated with AD-mediated itch232.

Taken together, TNF-/- mice have increased mast cell numbers in lesional skin

which correlate with clinical severity and TSLP mRNA levels. Anti- cKit improved the

development of AD and reduced TEWL in TNF-/- mice compared to the controls.

BMcMcs data indicate that MCs are not producing TSLP as this is rather produced

by keratinocytes. In search of MCs mediators which enhance TSLP levels in TNF-/-

mice, we identified that supernatants from resting MCs increased TSLP production

in skin biopsies. Histamine was not able to modulate TSLP production whereas

mMCP6 was significantly able to induce TSLP production by keratinocytes in skin

biopsies, indicating that tryptase might be the relevant factor involved in instructing

KCs to produce TSLP under TNF deficiency.

4.5 CONCLUSION AND OUTLOOK

The epidermis is a rigid layer of the skin protecting an organism against external

insults. It is also the anatomical structure to provide the skin barrier. An alteration of 84

Discussion

the skin barrier initiates inflammatory processes which may lead to skin diseases. In

this thesis it has been investigated how TSLP is regulated either after exposure to

external irritants or in AD; a chronic inflammatory skin disease.

The data show that TSLP was rapidly induced in keratinocytes upon irritant

exposure. Moreover skin perturbation of different kinds led to TSLP production

starting from injury to chemical exposure. These data suggest that TSLP is one of

the alarm signals in the skin upon exposure to any trauma. The mechanistic

analysis revealed that though exogenous TNF-α was capable of inducing TSLP in

vitro or ex vivo, endogenous TNF-α failed to do so. IL-1 a well known responder

upon irritation, was partially involved in SDS mediated TSLP production. As the

cascade of TSLP regulation and its role in irritation is still not very clear, further

extensive work is required to pin point the different factors involved. To this end,

skin biopsies and keratinocytes will be treated with different inhibitors or neutralizing

antibodies or agonists and their antagonists.

To better understand the role of TNF-α in AD, an allergen dependent mouse

dermatitis model was used. It showed an increased AD severity in TNF-/- compared

to wt mice. Further analysis of these mice including the skin and serum revealed no

major alterations of single cell types or factors except the TSLP expression locally

and systemically and increased mast cell numbers in the skin which correlated with

the clinical severity. As TNF-/- mice expressed more TSLP, it was important to

understand the role of TSLP for the progression of AD. To achieve this goal, anti-

TSLP was administered to neutralize the TSLP mediated effects in eczema. Such

treated mice showed a pronounced improvement of the AD, including a reduction of

the TEWL. These findings indicate that TSLP is most likely a key cytokine in severe

AD development under TNF deficiency. These data should be confirmed using TNF-

/-TSLP-/- double knockout mice to prove the role of TSLP in this scenario.

We observed an increase of the mast cell frequency which correlates with the

symptom score and TSLP expression. The application of an anti cKit to the TNF-/-

mice showed a reduction of eczema severity, indicating that mast cells are involved

in AD in this model. 85

Discussion

As previous data pointed out the role of mast cells in AD progression, the question

arose whether mast cells can directly induce TSLP as they are known to produce

TSLP210 or whether they can instruct keratinocytes to produce TSLP during AD

progression as they are also known to instruct epithelial cells to produce TSLP133. In

our hand data from BMcMcs have suggested that mast cells are not able to produce

TSLP upon activation with anti IgE in combination with different proinflammatory

cytokines. These results suggest that mast cells can instruct KCs to produce TSLP

in TNF-/- mice during AD development. To confirm this hypothesis, we stimulated

the skin biopsies with mast cell supernatants from stimulated and resting mast cells.

Supernatants from stimulated mast cells show no effect whereas the supernatants

from resting mast cells were capable to stimulate the cells from skin biopsies to

produce TSLP at significant levels. Next we tried to investigate which mast cell

mediator is responsible for the instruction of KCs to produce TSLP. Surprisingly

‘histamine’ the most abundant mast cell mediator was not able to induce TSLP

production. By contrast, we detected a significant increase of TSLP expression

upon treatment with the mast cell protease “mMCP6”. In the future it needs to be

confirmed whether tryptase is the main component of the MC supernatant triggering

TSLP production in the skin. Such conformation would be possible if mMCP6-/- mice

will be used in further experiments. Finally to confirm that mast cells are playing a

role by instructing KCs to produce TSLP in enhancement of AD under TNF

deficiency we need to perform the AD patch-model in mMCP6-/-TNF-/-TSLPR-/- triple

knockout mice.

Irritation and inflammation of the skin is a complex process including the interplay of

resident skin cells like KC and MC. Both TNF-α and TSLP contribute to this

interplay in this scenario with a significant extend. Mouse skin models can help to

better understand this complex network by applying knockout mice models or

targeting key effector mediator in appropriate disease models.

86

References

REFERENCES

1 Proksch E, Brandner JM, Jensen JM. The skin: an indispensable barrier. Exp Dermatol 2008; 17: 1063-72. 2 Oyoshi MK, Murphy GF, Geha RS. Filaggrin-deficient mice exhibit TH17-dominated skin inflammation and permissiveness to epicutaneous sensitization with protein antigen. J Allergy Clin Immunol 2009; 124: 485-93, 93 e1. 3 Hanel KH, Cornelissen C, Luscher B et al. Cytokines and the skin barrier. International journal of molecular sciences 2013; 14: 6720-45. 4 Fuchs E. Skin stem cells: rising to the surface. The Journal of cell biology 2008; 180: 273-84. 5 Kirschner N, Brandner JM. Barriers and more: functions of tight junction proteins in the skin. Ann N Y Acad Sci 2012; 1257: 158-66. 6 Hildenbrand M, Rhiemeier V, Hartenstein B et al. Impaired skin regeneration and remodeling after cutaneous injury and chemically induced hyperplasia in taps-transgenic mice. J Invest Dermatol 2010; 130: 1922-30. 7 Madison KC. Barrier function of the skin: "la raison d'etre" of the epidermis. J Invest Dermatol 2003; 121: 231-41. 8 Richardson M. Understanding the structure and function of the skin. Nursing times 2003; 99: 46-8. 9 Janetta Bensouilah PB. Aromadermatology: Aromatherapy in the Treatment and Care of Common Skin Conditions. 2006. 10 Janetta Bensouilah, Buck P. Aromadermatology: Aromatherapy in the Treatment and Care of Common Skin Conditions. Aromadermatology 2006. 11 Kanitakis J. Anatomy, histology and immunohistochemistry of normal human skin. European journal of dermatology : EJD 2002; 12: 390-9; quiz 400-1. 12 Bruce Alberts AJ, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular Biology of the Cell. Garland Science 2002; 4. 13 Flour M. The pathophysiology of vulnerable skin 2009. 14 Nestle FO, Di Meglio P, Qin JZ et al. Skin immune sentinels in health and disease. Nature reviews. Immunology 2009; 9: 679-91. 15 Angelova-Fischer I, Dapic I, Hoek AK et al. Skin Barrier Integrity and Natural Moisturising Factor Levels After Cumulative Dermal Exposure to Alkaline Agents in Atopic Dermatitis. Acta dermato-venereologica 2014. 16 Vinay Kumar AKA, Jon C. Aster. Robbins Basic Pathology. Elsevier Health Sciences 2012. 17 Le M, Schalkwijk J, Siegenthaler G et al. Changes in keratinocyte differentiation following mild irritation by sodium dodecyl sulphate. Archives of dermatological research 1996; 288: 684-90. 18 Varani J, Perone P, Spahlinger DM et al. Human skin in organ culture and human skin cells (keratinocytes and fibroblasts) in monolayer culture for assessment of chemically induced skin damage. Toxicologic pathology 2007; 35: 693-701. 19 Willis CM, Stephens CJ, Wilkinson JD. Differential patterns of epidermal leukocyte infiltration in patch test reactions to structurally unrelated chemical irritants. J Invest Dermatol 1993; 101: 364-70. 20 Jacobs JJ, Lehe CL, Hasegawa H et al. Skin irritants and contact sensitizers induce Langerhans cell migration and maturation at irritant concentration. Exp Dermatol 2006; 15: 432-40. 21 Escobar-Chavez JJ, Merino-Sanjuan V, Lopez-Cervantes M et al. The tape-stripping technique as a method for drug quantification in skin. Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques 2008; 11: 104-30.

87

References

22 Nickoloff BJ, Naidu Y. Perturbation of epidermal barrier function correlates with initiation of cytokine cascade in human skin. Journal of the American Academy of Dermatology 1994; 30: 535-46. 23 Wilmer JL, Burleson FG, Kayama F et al. Cytokine induction in human epidermal keratinocytes exposed to contact irritants and its relation to chemical-induced inflammation in mouse skin. J Invest Dermatol 1994; 102: 915-22. 24 Usatine RP, Riojas M. Diagnosis and management of contact dermatitis. American family physician 2010; 82: 249-55. 25 Coenraads PJ, Goncalo M. Skin diseases with high public health impact. Contact dermatitis. European journal of dermatology : EJD 2007; 17: 564-5. 26 Nosbaum A, Vocanson M, Rozieres A et al. Allergic and irritant contact dermatitis. European journal of dermatology : EJD 2009; 19: 325-32. 27 Slodownik D, Lee A, Nixon R. Irritant contact dermatitis: a review. The Australasian journal of dermatology 2008; 49: 1-9; quiz 10-1. 28 Dhingra N, Gulati N, Guttman-Yassky E. Mechanisms of contact sensitization offer insights into the role of barrier defects vs. intrinsic immune abnormalities as drivers of atopic dermatitis. J Invest Dermatol 2013; 133: 2311-4. 29 Vocanson M, Hennino A, Chavagnac C et al. Contribution of CD4(+ )and CD8(+) T-cells in contact hypersensitivity and allergic contact dermatitis. Expert Rev Clin Immunol 2005; 1: 75-86. 30 Khuntia A, Baldwin J. Contact Dermatitis. 2004. 31 Bieber T. Atopic dermatitis. The New England journal of medicine 2008; 358: 1483-94. 32 Leung DY, Boguniewicz M, Howell MD et al. New insights into atopic dermatitis. The Journal of clinical investigation 2004; 113: 651-7. 33 Worm M, Forschner K, Lee HH et al. Frequency of atopic dermatitis and relevance of food allergy in adults in Germany. Acta dermato-venereologica 2006; 86: 119-22. 34 Wuthrich B, Cozzio A, Roll A et al. Atopic eczema: genetics or environment? Annals of agricultural and environmental medicine : AAEM 2007; 14: 195-201. 35 Maintz L, Novak N. Getting more and more complex: the pathophysiology of atopic eczema. European journal of dermatology : EJD 2007; 17: 267-83. 36 Oyoshi MK, He R, Kumar L et al. Cellular and molecular mechanisms in atopic dermatitis. Advances in immunology 2009; 102: 135-226. 37 Bieber T. Atopic dermatitis. Annals of dermatology 2010; 22: 125-37. 38 Ong PY. New insights in the pathogenesis of atopic dermatitis. Pediatric research 2014; 75: 171-5. 39 Kuo IH, Yoshida T, De Benedetto A et al. The cutaneous innate immune response in patients with atopic dermatitis. J Allergy Clin Immunol 2013; 131: 266-78. 40 Chan LS. Atopic dermatitis in 2008. Current directions in autoimmunity 2008; 10: 76-118. 41 Bohme M, Soderhall C, Kull I et al. Filaggrin mutations increase the risk for persistent dry skin and eczema independent of sensitization. J Allergy Clin Immunol 2012; 129: 1153-5. 42 Howell MD, Kim BE, Gao P et al. Cytokine modulation of atopic dermatitis filaggrin skin expression. J Allergy Clin Immunol 2007; 120: 150-5. 43 Thyssen JP, Linneberg A, Carlsen BC et al. A possible association between a dysfunctional skin barrier (filaggrin null-mutation status) and diabetes: a cross-sectional study. BMJ open 2011; 1: e000062. 44 Brandt EB, Sivaprasad U. Th2 Cytokines and Atopic Dermatitis. Journal of clinical & cellular immunology 2011; 2. 45 Darsow U, Forer I, Ring J. Allergen-specific immunotherapy in atopic eczema. Current allergy and asthma reports 2011; 11: 277-83.

88

References

46 Jin H, He R, Oyoshi M et al. Animal models of atopic dermatitis. J Invest Dermatol 2009; 129: 31-40. 47 Gittler JK, Shemer A, Suarez-Farinas M et al. Progressive activation of T(H)2/T(H)22 cytokines and selective epidermal proteins characterizes acute and chronic atopic dermatitis. J Allergy Clin Immunol 2012; 130: 1344-54. 48 Nakajima S, Kitoh A, Egawa G et al. IL-17A as an inducer for Th2 immune responses in murine atopic dermatitis models. J Invest Dermatol 2014; 134: 2122-30. 49 Yoo J, Omori M, Gyarmati D et al. Spontaneous atopic dermatitis in mice expressing an inducible thymic stromal lymphopoietin transgene specifically in the skin. J Exp Med 2005; 202: 541-9. 50 Li M, Hener P, Zhang Z et al. Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis. Proc Natl Acad Sci U S A 2006; 103: 11736-41. 51 Briot A, Deraison C, Lacroix M et al. Kallikrein 5 induces atopic dermatitis-like lesions through PAR2-mediated thymic stromal lymphopoietin expression in Netherton syndrome. J Exp Med 2009; 206: 1135-47. 52 Gutermuth J, Ollert M, Ring J et al. Mouse models of atopic eczema critically evaluated. International archives of allergy and immunology 2004; 135: 262-76. 53 Spergel JM, Mizoguchi E, Oettgen H et al. Roles of TH1 and TH2 cytokines in a murine model of allergic dermatitis. The Journal of clinical investigation 1999; 103: 1103-11. 54 Niebuhr M, Werfel T. Innate immunity, allergy and atopic dermatitis. Current opinion in allergy and clinical immunology 2010; 10: 463-8. 55 Eckert RL, Rorke EA. Molecular biology of keratinocyte differentiation. Environmental health perspectives 1989; 80: 109-16. 56 Barker JN, Mitra RS, Griffiths CE et al. Keratinocytes as initiators of inflammation. Lancet 1991; 337: 211-4. 57 Palmer CN, Irvine AD, Terron-Kwiatkowski A et al. Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis. Nature genetics 2006; 38: 441-6. 58 De Benedetto A, Rafaels NM, McGirt LY et al. Tight junction defects in patients with atopic dermatitis. J Allergy Clin Immunol 2011; 127: 773-86 e1-7. 59 Gittler JK, Krueger JG, Guttman-Yassky E. Atopic dermatitis results in intrinsic barrier and immune abnormalities: implications for contact dermatitis. J Allergy Clin Immunol 2013; 131: 300-13. 60 He R, Geha RS. Thymic stromal lymphopoietin. Ann N Y Acad Sci 2010; 1183: 13-24. 61 Hoefakker S, Caubo M, van 't Erve EH et al. In vivo cytokine profiles in allergic and irritant contact dermatitis. Contact dermatitis 1995; 33: 258-66. 62 Wood LC, Elias PM, Calhoun C et al. Barrier disruption stimulates interleukin-1 alpha expression and release from a pre-formed pool in murine epidermis. J Invest Dermatol 1996; 106: 397-403. 63 Smith HR, Basketter DA, McFadden JP. Irritant dermatitis, irritancy and its role in allergic contact dermatitis. Clinical and experimental dermatology 2002; 27: 138-46. 64 Grangsjo A, Leijon-Kuligowski A, Torma H et al. Different pathways in irritant contact eczema? Early differences in the epidermal elemental content and expression of cytokines after application of 2 different irritants. Contact dermatitis 1996; 35: 355-60. 65 de Jongh CM, Lutter R, Verberk MM et al. Differential cytokine expression in skin after single and repeated irritation by sodium lauryl sulphate. Exp Dermatol 2007; 16: 1032-40. 66 McKenzie RC, Sauder DN. The role of keratinocyte cytokines in inflammation and immunity. J Invest Dermatol 1990; 95: 105S-7S. 67 Lee HY, Stieger M, Yawalkar N et al. Cytokines and chemokines in irritant contact dermatitis. Mediators of inflammation 2013; 2013: 916497. 68 Leung DY, Bieber T. Atopic dermatitis. Lancet 2003; 361: 151-60.

89

References

69 Kasraie S, Werfel T. Role of macrophages in the pathogenesis of atopic dermatitis. Mediators of inflammation 2013; 2013: 942375. 70 Irvine AD, McLean WH. Breaking the (un)sound barrier: filaggrin is a major gene for atopic dermatitis. J Invest Dermatol 2006; 126: 1200-2. 71 Akdis CA, Akdis M, Bieber T et al. Diagnosis and treatment of atopic dermatitis in children and adults: European Academy of Allergology and Clinical Immunology/American Academy of Allergy, Asthma and Immunology/PRACTALL Consensus Report. J Allergy Clin Immunol 2006; 118: 152-69. 72 Ong PY, Leung DY. Atopic dermatitis. Clinical allergy and immunology 2002; 16: 355-79. 73 de Koning HD, Kamsteeg M, Rodijk-Olthuis D et al. Epidermal expression of host response genes upon skin barrier disruption in normal skin and uninvolved skin of psoriasis and atopic dermatitis patients. J Invest Dermatol 2011; 131: 263-6. 74 Jessup HK, Brewer AW, Omori M et al. Intradermal administration of thymic stromal lymphopoietin induces a T cell- and eosinophil-dependent systemic Th2 inflammatory response. J Immunol 2008; 181: 4311-9. 75 Nedospasov SA, Hirt B, Shakhov AN et al. The genes for tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) are tandemly arranged on chromosome 17 of the mouse. Nucleic acids research 1986; 14: 7713-25. 76 Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell death and differentiation 2003; 10: 45-65. 77 Palladino MA, Bahjat FR, Theodorakis EA et al. Anti-TNF-alpha therapies: the next generation. Nature reviews. Drug discovery 2003; 2: 736-46. 78 Bradley JR. TNF-mediated inflammatory disease. The Journal of pathology 2008; 214: 149-60. 79 Eigler A, Sinha B, Hartmann G et al. Taming TNF: strategies to restrain this proinflammatory cytokine. Immunology today 1997; 18: 487-92. 80 Kruglov AA, Kuchmiy A, Grivennikov SI et al. Physiological functions of tumor necrosis factor and the consequences of its pathologic overexpression or blockade: mouse models. Cytokine & growth factor reviews 2008; 19: 231-44. 81 Taylor PC, Feldmann M. Anti-TNF biologic agents: still the therapy of choice for rheumatoid arthritis. Nature reviews. Rheumatology 2009; 5: 578-82. 82 Lewis RW, McCall JC, Botham PA et al. Investigation of TNF-alpha release as a measure of skin irritancy. Toxicol In Vitro 1993; 7: 393-5. 83 Liao F, Rabin RL, Smith CS et al. CC-chemokine receptor 6 is expressed on diverse memory subsets of T cells and determines responsiveness to macrophage inflammatory protein 3 alpha. J Immunol 1999; 162: 186-94. 84 Corsini E, Terzoli A, Bruccoleri A et al. Induction of tumor necrosis factor-alpha in vivo by a skin irritant, tributyltin, through activation of transcription factors: its pharmacological modulation by anti-inflammatory drugs. J Invest Dermatol 1997; 108: 892-6. 85 Piguet PF, Grau GE, Hauser C et al. Tumor necrosis factor is a critical mediator in hapten induced irritant and contact hypersensitivity reactions. J Exp Med 1991; 173: 673-9. 86 Takahashi T, Sasaki Y, Hama K et al. Production of IL-4, IL-2, IFN-gamma, and TNF-alpha by peripheral blood mononuclear cells of patients with atopic dermatitis. J Dermatol Sci 1992; 3: 172-80. 87 Poulsen LK, Bindslev-Jensen C, Diamant M et al. Biomolecular regulation of the IgE immune response III. Cytokine profiles in atopic dermatitis, inhalant allergy and non-allergic donors. Cytokine 1996; 8: 651-7. 88 Jeong CW, Ahn KS, Rho NK et al. Differential in vivo cytokine mRNA expression in lesional skin of intrinsic vs. extrinsic atopic dermatitis patients using semiquantitative RT-PCR. Clin Exp Allergy 2003; 33: 1717-24.

90

References

89 Nomura I, Goleva E, Howell MD et al. Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes. J Immunol 2003; 171: 3262-9. 90 Lee HH, Song IH, Friedrich M et al. Cutaneous side-effects in patients with rheumatic diseases during application of tumour necrosis factor-alpha antagonists. The British journal of dermatology 2007; 156: 486-91. 91 Esmailzadeh A, Yousefi P, Farhi D et al. Predictive factors of eczema-like eruptions among patients without cutaneous psoriasis receiving infliximab: a cohort study of 92 patients. Dermatology 2009; 219: 263-7. 92 Rashmi Sharma CLS. TNF-alpha inhibitors: Current indications. Indian Journal of Critical Care Medicine 2007; 11: 139-48 93 Chen X, Baumel M, Mannel DN et al. Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells. J Immunol 2007; 179: 154-61. 94 Kassiotis G, Kollias G. Uncoupling the proinflammatory from the immunosuppressive properties of tumor necrosis factor (TNF) at the p55 TNF receptor level: implications for pathogenesis and therapy of autoimmune demyelination. J Exp Med 2001; 193: 427-34. 95 Noti M, Corazza N, Mueller C et al. TNF suppresses acute intestinal inflammation by inducing local glucocorticoid synthesis. J Exp Med 2010; 207: 1057-66. 96 Friend SL, Hosier S, Nelson A et al. A thymic stromal cell line supports in vitro development of surface IgM+ B cells and produces a novel growth factor affecting B and T lineage cells. Experimental hematology 1994; 22: 321-8. 97 Sims JE, Williams DE, Morrissey PJ et al. Molecular cloning and biological characterization of a novel murine lymphoid growth factor. J Exp Med 2000; 192: 671-80. 98 Pandey A, Ozaki K, Baumann H et al. Cloning of a receptor subunit required for signaling by thymic stromal lymphopoietin. Nat Immunol 2000; 1: 59-64. 99 Park LS, Martin U, Garka K et al. Cloning of the murine thymic stromal lymphopoietin (TSLP) receptor: Formation of a functional heteromeric complex requires interleukin 7 receptor. J Exp Med 2000; 192: 659-70. 100 Quentmeier H, Drexler HG, Fleckenstein D et al. Cloning of human thymic stromal lymphopoietin (TSLP) and signaling mechanisms leading to proliferation. Leukemia 2001; 15: 1286-92. 101 Reche PA, Soumelis V, Gorman DM et al. Human thymic stromal lymphopoietin preferentially stimulates myeloid cells. J Immunol 2001; 167: 336-43. 102 Rimoldi M, Chieppa M, Salucci V et al. Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells. Nat Immunol 2005; 6: 507-14. 103 Soumelis V, Reche PA, Kanzler H et al. Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP. Nat Immunol 2002; 3: 673-80. 104 Watanabe N, Wang YH, Lee HK et al. Hassall's corpuscles instruct dendritic cells to induce CD4+CD25+ regulatory T cells in human thymus. Nature 2005; 436: 1181-5. 105 Isaksen DE, Baumann H, Trobridge PA et al. Requirement for stat5 in thymic stromal lymphopoietin-mediated signal transduction. J Immunol 1999; 163: 5971-7. 106 Arima K, Watanabe N, Hanabuchi S et al. Distinct signal codes generate dendritic cell functional plasticity. Science signaling 2010; 3: ra4. 107 Wohlmann A, Sebastian K, Borowski A et al. Signal transduction by the atopy-associated human thymic stromal lymphopoietin (TSLP) receptor depends on Janus kinase function. Biological chemistry 2010; 391: 181-6. 108 Zhong J, Kim MS, Chaerkady R et al. TSLP signaling network revealed by SILAC-based phosphoproteomics. Molecular & cellular proteomics : MCP 2012; 11: M112 017764. 109 Taylor BC, Zaph C, Troy AE et al. TSLP regulates intestinal immunity and inflammation in mouse models of helminth infection and colitis. J Exp Med 2009; 206: 655-67.

91

References

110 Olkhanud PB, Rochman Y, Bodogai M et al. Thymic stromal lymphopoietin is a key mediator of breast cancer progression. J Immunol 2011; 186: 5656-62. 111 Roan F, Bell BD, Stoklasek TA et al. The multiple facets of thymic stromal lymphopoietin (TSLP) during allergic inflammation and beyond. Journal of leukocyte biology 2012; 91: 877-86. 112 West EE, Kashyap M, Leonard WJ. TSLP: A Key Regulator of Asthma Pathogenesis. Drug discovery today. Disease mechanisms 2012; 9. 113 Zhou B, Comeau MR, De Smedt T et al. Thymic stromal lymphopoietin as a key initiator of allergic airway inflammation in mice. Nat Immunol 2005; 6: 1047-53. 114 Al-Shami A, Spolski R, Kelly J et al. A role for TSLP in the development of inflammation in an asthma model. J Exp Med 2005; 202: 829-39. 115 He R, Oyoshi MK, Garibyan L et al. TSLP acts on infiltrating effector T cells to drive allergic skin inflammation. Proc Natl Acad Sci U S A 2008; 105: 11875-80. 116 Elias PM. Stratum corneum defensive functions: an integrated view. J Invest Dermatol 2005; 125: 183-200. 117 Angelova-Fischer I, Fernandez IM, Donnadieu MH et al. Injury to the stratum corneum induces in vivo expression of human thymic stromal lymphopoietin in the epidermis. J Invest Dermatol 2010; 130: 2505-7. 118 Elias PM, Steinhoff M. "Outside-to-inside" (and now back to "outside") pathogenic mechanisms in atopic dermatitis. J Invest Dermatol 2008; 128: 1067-70. 119 Elias PM, Wood LC, Feingold KR. Epidermal pathogenesis of inflammatory dermatoses. American journal of contact dermatitis : official journal of the American Contact Dermatitis Society 1999; 10: 119-26. 120 Lee HC, Headley MB, Iseki M et al. Cutting edge: Inhibition of NF-kappaB-mediated TSLP expression by retinoid X receptor. J Immunol 2008; 181: 5189-93. 121 Liu YJ. Thymic stromal lymphopoietin: master switch for allergic inflammation. J Exp Med 2006; 203: 269-73. 122 Allakhverdi Z, Comeau MR, Jessup HK et al. Thymic stromal lymphopoietin is released by human epithelial cells in response to microbes, trauma, or inflammation and potently activates mast cells. J Exp Med 2007; 204: 253-8. 123 Bogiatzi SI, Fernandez I, Bichet JC et al. Cutting Edge: Proinflammatory and Th2 cytokines synergize to induce thymic stromal lymphopoietin production by human skin keratinocytes. J Immunol 2007; 178: 3373-7. 124 Liu YJ. Thymic stromal lymphopoietin and OX40 ligand pathway in the initiation of dendritic cell-mediated allergic inflammation. J Allergy Clin Immunol 2007; 120: 238-44; quiz 45-6. 125 Demehri S, Liu Z, Lee J et al. Notch-deficient skin induces a lethal systemic B-lymphoproliferative disorder by secreting TSLP, a sentinel for epidermal integrity. PLoS Biol 2008; 6: e123. 126 Demehri S, Morimoto M, Holtzman MJ et al. Skin-derived TSLP triggers progression from epidermal-barrier defects to asthma. PLoS Biol 2009; 7: e1000067. 127 Ziegler SF, Artis D. Sensing the outside world: TSLP regulates barrier immunity. Nat Immunol 2010; 11: 289-93. 128 Zhang Z, Hener P, Frossard N et al. Thymic stromal lymphopoietin overproduced by keratinocytes in mouse skin aggravates experimental asthma. Proc Natl Acad Sci U S A 2009; 106: 1536-41. 129 Miazgowicz MM, Headley MB, Larson RP et al. Thymic stromal lymphopoietin and the pathophysiology of atopic disease. Expert Rev Clin Immunol 2009; 5: 547-56. 130 Li M, Hener P, Zhang Z et al. Induction of thymic stromal lymphopoietin expression in keratinocytes is necessary for generating an atopic dermatitis upon application of the active vitamin D3 analogue MC903 on mouse skin. J Invest Dermatol 2009; 129: 498-502.

92

References

131 Pasparakis M, Alexopoulou L, Episkopou V et al. Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J Exp Med 1996; 184: 1397-411. 132 Dahten A, Koch C, Ernst D et al. Systemic PPARgamma ligation inhibits allergic immune response in the skin. J Invest Dermatol 2008; 128: 2211-8. 133 Miyata M, Hatsushika K, Ando T et al. Mast cell regulation of epithelial TSLP expression plays an important role in the development of allergic rhinitis. European journal of immunology 2008; 38: 1487-92. 134 Brandt EB, Strait RT, Hershko D et al. Mast cells are required for experimental oral allergen-induced diarrhea. The Journal of clinical investigation 2003; 112: 1666-77. 135 Jahnz-Rozyk K, Targowski T, Paluchowska E et al. Serum thymus and activation-regulated chemokine, macrophage-derived chemokine and eotaxin as markers of severity of atopic dermatitis. Allergy 2005; 60: 685-8. 136 Steiner M, Aikman-Green S, Prescott GJ et al. Side-by-side comparison of an open-chamber (TM 300) and a closed-chamber (Vapometer) transepidermal water loss meter. Skin research and technology : official journal of International Society for Bioengineering and the Skin 2011; 17: 366-72. 137 Redvers RP, Kaur P. Serial cultivation of primary adult murine keratinocytes. Methods Mol Biol 2005; 289: 15-22. 138 Artuc M, Steckelings UM, Grutzkau A et al. A long-term coculture model for the study of mast cell-keratinocyte interactions. J Invest Dermatol 2002; 119: 411-5. 139 Byrne AM, Goleva E, Chouiali F et al. Induction of GITRL expression in human keratinocytes by Th2 cytokines and TNF-alpha: implications for atopic dermatitis. Clin Exp Allergy 2012; 42: 550-9. 140 Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nature protocols 2008; 3: 1101-8. 141 Mrabet-Dahbi S, Metz M, Dudeck A et al. Murine mast cells secrete a unique profile of cytokines and prostaglandins in response to distinct TLR2 ligands. Exp Dermatol 2009; 18: 437-44. 142 Nakahata T, Spicer SS, Cantey JR et al. Clonal assay of mouse mast cell colonies in methylcellulose culture. Blood 1982; 60: 352-61. 143 Ogawa M, Matsuzaki Y, Nishikawa S et al. Expression and function of c-kit in hemopoietic progenitor cells. J Exp Med 1991; 174: 63-71. 144 MacNeil AJ, Yang YJ, Lin TJ. MAPK kinase 3 specifically regulates Fc epsilonRI-mediated IL-4 production by mast cells. J Immunol 2011; 187: 3374-82. 145 Tanaka A, Matsuda H. IgE crosslinkage of Fcepsilon receptor I induces both production and activation of matrix metalloproteinase-9 in mast cells. Cellular immunology 2004; 228: 66-75. 146 Weber S, Niessen MT, Prox J et al. The disintegrin/metalloproteinase Adam10 is essential for epidermal integrity and Notch-mediated signaling. Development 2011; 138: 495-505. 147 Bertolini TM, Giorgione J, Harvey DF et al. Protein kinase C translocation by modified phorbol esters with functionalized lipophilic regions. J Org Chem 2003; 68: 5028-36. 148 Kato A, Favoreto S, Jr., Avila PC et al. TLR3- and Th2 cytokine-dependent production of thymic stromal lymphopoietin in human airway epithelial cells. J Immunol 2007; 179: 1080-7. 149 Kinoshita H, Takai T, Le TA et al. Cytokine milieu modulates release of thymic stromal lymphopoietin from human keratinocytes stimulated with double-stranded RNA. J Allergy Clin Immunol 2009; 123: 179-86. 150 Lee HC, Ziegler SF. Inducible expression of the proallergic cytokine thymic stromal lymphopoietin in airway epithelial cells is controlled by NFkappaB. Proc Natl Acad Sci U S A 2007; 104: 914-9.

93

References

151 Saenz SA, Taylor BC, Artis D. Welcome to the neighborhood: epithelial cell-derived cytokines license innate and adaptive immune responses at mucosal sites. Immunol Rev 2008; 226: 172-90. 152 Nonaka M, Fukumoto A, Ogihara N et al. Synergistic induction of thymic stromal lymphopoietin by tumor necrosis factor alpha and Th2 cytokine in nasal polyp fibroblasts. Am J Rhinol Allergy 2010; 24: e14-8. 153 Xie Y, Takai T, Chen X et al. Long TSLP transcript expression and release of TSLP induced by TLR ligands and cytokines in human keratinocytes. J Dermatol Sci 2012; 66: 233-7. 154 Mudnakudu Nagaraju KK, Babina M, Worm M. Opposing effects on immune function and skin barrier regulation by the proteasome inhibitor bortezomib in an allergen-induced eczema model. Exp Dermatol 2013; 22: 742-7. 155 Cohen C, Dossou G, Rougier A et al. Measurement of inflammatory mediators produced by human keratinocytes in vitro: A predictive assessment of cutaneous irritation. Toxicol In Vitro 1991; 5: 407-10. 156 Spiekstra SW, Toebak MJ, Sampat-Sardjoepersad S et al. Induction of cytokine (interleukin-1alpha and tumor necrosis factor-alpha) and chemokine (CCL20, CCL27, and CXCL8) alarm signals after allergen and irritant exposure. Exp Dermatol 2005; 14: 109-16. 157 Warner RR, Boissy YL, Lilly NA et al. Water disrupts stratum corneum lipid lamellae: damage is similar to surfactants. J Invest Dermatol 1999; 113: 960-6. 158 Pessolano LG, Jr., Sullivan CP, Seidl SE et al. Trafficking of endogenous smooth muscle cell cholesterol: a role for serum amyloid A and interleukin-1beta. Arterioscler Thromb Vasc Biol 2012; 32: 2741-50. 159 Welker P, Grabbe J, Gibbs B et al. Nerve growth factor-beta induces mast-cell marker expression during in vitro culture of human umbilical cord blood cells. Immunology 2000; 99: 418-26. 160 Wang YH, Liu YJ. OX40-OX40L interactions: a promising therapeutic target for allergic diseases? The Journal of clinical investigation 2007; 117: 3655-7. 161 Urb M, Sheppard DC. The role of mast cells in the defence against pathogens. PLoS pathogens 2012; 8: e1002619. 162 Kawakami T, Ando T, Kimura M et al. Mast cells in atopic dermatitis. Current opinion in immunology 2009; 21: 666-78. 163 Danso MO, van Drongelen V, Mulder A et al. TNF-alpha and Th2 cytokines induce atopic dermatitis-like features on epidermal differentiation proteins and stratum corneum lipids in human skin equivalents. J Invest Dermatol 2014; 134: 1941-50. 164 Takai T. TSLP expression: cellular sources, triggers, and regulatory mechanisms. Allergol Int 2012; 61: 3-17. 165 Murthy A, Shao YW, Narala SR et al. Notch activation by the metalloproteinase ADAM17 regulates myeloproliferation and atopic barrier immunity by suppressing epithelial cytokine synthesis. Immunity 2012; 36: 105-19. 166 Tsai M, Chen CC, Mukai K et al. Thymic stromal lymphopoietin contributes to myeloid hyperplasia and increased immunoglobulins, but not epidermal hyperplasia, in RabGEF1-deficient mice. Am J Pathol 2010; 177: 2411-20. 167 Wang Z, Zhang LJ, Guha G et al. Selective ablation of Ctip2/Bcl11b in epidermal keratinocytes triggers atopic dermatitis-like skin inflammatory responses in adult mice. PLoS One 2012; 7: e51262. 168 Lee CH, Maibach HI. The sodium lauryl sulfate model: an overview. Contact dermatitis 1995; 33: 1-7. 169 Dugard PH, Scheuplein RJ. Effects of ionic surfactants on the permeability of human epidermis: an electrometric study. J Invest Dermatol 1973; 60: 263-9. 170 Boukhatem MN, Kameli A, Ferhat MA et al. Rose geranium essential oil as a source of new and safe anti-inflammatory drugs. The Libyan journal of medicine 2013; 8: 22520.

94

References

171 Rauh V, Arunajadai S, Horton M et al. Seven-year neurodevelopmental scores and prenatal exposure to chlorpyrifos, a common agricultural pesticide. Environmental health perspectives 2011; 119: 1196-201. 172 Furstenberger G, Csuk-Glanzer BI, Marks F et al. Phorbol ester-induced leukotriene biosynthesis and tumor promotion in mouse epidermis. Carcinogenesis 1994; 15: 2823-7. 173 Flint MS, Miller DB, Tinkle SS. Restraint-induced modulation of allergic and irritant contact dermatitis in male and female B6.129 mice. Brain, behavior, and immunity 2000; 14: 256-69. 174 Terada M, Tsutsui H, Imai Y et al. Contribution of IL-18 to atopic-dermatitis-like skin inflammation induced by Staphylococcus aureus product in mice. Proc Natl Acad Sci U S A 2006; 103: 8816-21. 175 Demehri S, Turkoz A, Manivasagam S et al. Elevated epidermal thymic stromal lymphopoietin levels establish an antitumor environment in the skin. Cancer Cell 2012; 22: 494-505. 176 Di Piazza M, Nowell CS, Koch U et al. Loss of cutaneous TSLP-dependent immune responses skews the balance of inflammation from tumor protective to tumor promoting. Cancer Cell 2012; 22: 479-93. 177 Jang Y, Jeong SH, Park YH et al. UVB induces HIF-1alpha-dependent TSLP expression via the JNK and ERK pathways. J Invest Dermatol 2013; 133: 2601-8. 178 Oyoshi MK, Larson RP, Ziegler SF et al. Mechanical injury polarizes skin dendritic cells to elicit a T(H)2 response by inducing cutaneous thymic stromal lymphopoietin expression. J Allergy Clin Immunol 2010; 126: 976-84, 84 e1-5. 179 Sano Y, Masuda K, Tamagawa-Mineoka R et al. Thymic stromal lymphopoietin expression is increased in the horny layer of patients with atopic dermatitis. Clin Exp Immunol 2013; 171: 330-7. 180 Yockey LJ, Demehri S, Turkoz M et al. The absence of a microbiota enhances TSLP expression in mice with defective skin barrier but does not affect the severity of their allergic inflammation. J Invest Dermatol 2013; 133: 2714-21. 181 Carta S, Lavieri R, Rubartelli A. Different Members of the IL-1 Family Come Out in Different Ways: DAMPs vs. Cytokines? Front Immunol 2013; 4: 123. 182 van de Veerdonk FL, Netea MG. New Insights in the Immunobiology of IL-1 Family Members. Front Immunol 2013; 4: 167. 183 Cevikbas F, Steinhoff M. IL-33: a novel danger signal system in atopic dermatitis. J Invest Dermatol 2012; 132: 1326-9. 184 Keyel PA. How is inflammation initiated? Individual influences of IL-1, IL-18 and HMGB1. Cytokine 2014. 185 Leonard WJ. TSLP: finally in the limelight. Nat Immunol 2002; 3: 605-7. 186 Lin Q, Wang L, Lin Y et al. Toll-like receptor 3 ligand polyinosinic:polycytidylic acid promotes wound healing in human and murine skin. J Invest Dermatol 2012; 132: 2085-92. 187 Kamekura R, Kojima T, Koizumi J et al. Thymic stromal lymphopoietin enhances tight-junction barrier function of human nasal epithelial cells. Cell Tissue Res 2009; 338: 283-93. 188 Nishiura H, Kido M, Aoki N et al. Increased susceptibility to autoimmune gastritis in thymic stromal lymphopoietin receptor-deficient mice. J Immunol 2012; 188: 190-7. 189 Reardon C, Lechmann M, Brustle A et al. Thymic stromal lymphopoetin-induced expression of the endogenous inhibitory enzyme SLPI mediates recovery from colonic inflammation. Immunity 2011; 35: 223-35. 190 Sonesson A, Kasetty G, Olin AI et al. Thymic stromal lymphopoietin exerts antimicrobial activities. Exp Dermatol 2011; 20: 1004-10. 191 Siracusa MC, Saenz SA, Wojno ED et al. Thymic stromal lymphopoietin-mediated extramedullary hematopoiesis promotes allergic inflammation. Immunity 2013; 39: 1158-70. 192 Leung DY. Atopic dermatitis: new insights and opportunities for therapeutic intervention. J Allergy Clin Immunol 2000; 105: 860-76.

95

References

193 Prodanovich S, Ricotti C, Glick BP et al. Etanercept: an evolving role in psoriasis and psoriatic arthritis. American journal of clinical dermatology 2010; 11 Suppl 1: 3-9. 194 Youdim A, Vasiliauskas EA, Targan SR et al. A pilot study of adalimumab in infliximab-allergic patients. Inflammatory bowel diseases 2004; 10: 333-8. 195 Guttman-Yassky E, Lowes MA, Fuentes-Duculan J et al. Major differences in inflammatory dendritic cells and their products distinguish atopic dermatitis from psoriasis. J Allergy Clin Immunol 2007; 119: 1210-7. 196 Chan JL, Davis-Reed L, Kimball AB. Counter-regulatory balance: atopic dermatitis in patients undergoing infliximab infusion therapy. Journal of drugs in dermatology : JDD 2004; 3: 315-8. 197 Lai-Cheong J, Warren R, Bucknall R et al. Etanercept-induced dermatitis in a patient with rheumatoid arthritis. Journal of the European Academy of Dermatology and Venereology : JEADV 2006; 20: 614-5. 198 Mangge H, Gindl S, Kenzian H et al. Atopic dermatitis as a side effect of anti-tumor necrosis factor-alpha therapy. The Journal of rheumatology 2003; 30: 2506-7. 199 Choi YJ, Lee HJ, Lee do H et al. Therapeutic effects and immunomodulation of suanbo mineral water therapy in a murine model of atopic dermatitis. Annals of dermatology 2013; 25: 462-70. 200 Woodward AL, Spergel JM, Alenius H et al. An obligate role for T-cell receptor alphabeta+ T cells but not T-cell receptor gammadelta+ T cells, B cells, or CD40/CD40L interactions in a mouse model of atopic dermatitis. J Allergy Clin Immunol 2001; 107: 359-66. 201 Hijnen D, Knol EF, Gent YY et al. CD8(+) T cells in the lesional skin of atopic dermatitis and psoriasis patients are an important source of IFN-gamma, IL-13, IL-17, and IL-22. J Invest Dermatol 2013; 133: 973-9. 202 Turner MJ, Travers JB, Kaplan MH. T helper cell subsets in the development of atopic dermatitis. Journal of drugs in dermatology : JDD 2012; 11: 1174-8. 203 Aioi A, Tonogaito H, Suto H et al. Impairment of skin barrier function in NC/Nga Tnd mice as a possible model for atopic dermatitis. The British journal of dermatology 2001; 144: 12-8. 204 Gupta J, Grube E, Ericksen MB et al. Intrinsically defective skin barrier function in children with atopic dermatitis correlates with disease severity. J Allergy Clin Immunol 2008; 121: 725-30 e2. 205 Ziegler SF, Roan F, Bell BD et al. The biology of thymic stromal lymphopoietin (TSLP). Advances in pharmacology 2013; 66: 129-55. 206 Le TA, Takai T, Vu AT et al. Flagellin induces the expression of thymic stromal lymphopoietin in human keratinocytes via toll-like receptor 5. International archives of allergy and immunology 2011; 155: 31-7. 207 Han NR, Oh HA, Nam SY et al. TSLP Induces Mast Cell Development and Aggravates Allergic Reactions through the Activation of MDM2 and STAT6. J Invest Dermatol 2014; 134: 2521-30. 208 Gao PS, Rafaels NM, Mu D et al. Genetic variants in thymic stromal lymphopoietin are associated with atopic dermatitis and eczema herpeticum. J Allergy Clin Immunol 2010; 125: 1403-7 e4. 209 He R, Kim HY, Yoon J et al. Exaggerated IL-17 response to epicutaneous sensitization mediates airway inflammation in the absence of IL-4 and IL-13. J Allergy Clin Immunol 2009; 124: 761-70 e1. 210 Okayama Y, Okumura S, Sagara H et al. FcepsilonRI-mediated thymic stromal lymphopoietin production by interleukin-4-primed human mast cells. The European respiratory journal 2009; 34: 425-35. 211 Ando T, Xiao W, Gao P et al. Critical role for mast cell Stat5 activity in skin inflammation. Cell reports 2014; 6: 366-76.

96

References

212 Sebastian K, Borowski A, Kuepper M et al. Signal transduction around thymic stromal lymphopoietin (TSLP) in atopic asthma. Cell communication and signaling : CCS 2008; 6: 5. 213 Kuehn HS, Radinger M, Gilfillan AM. Measuring mast cell mediator release. Current protocols in immunology / edited by John E. Coligan ... [et al.] 2010; Chapter 7: Unit7 38. 214 Reuter S, Stassen M, Taube C. Mast cells in allergic asthma and beyond. Yonsei medical journal 2010; 51: 797-807. 215 Boyce JA. Eicosanoid mediators of mast cells: receptors, regulation of synthesis, and pathobiologic implications. Chemical immunology and allergy 2005; 87: 59-79. 216 Gilfillan AM, Beaven MA. Regulation of mast cell responses in health and disease. Critical reviews in immunology 2011; 31: 475-529. 217 Katsanos GS, Anogeianaki A, Orso C et al. Mast cells and chemokines. Journal of biological regulators and homeostatic agents 2008; 22: 145-51. 218 McNeil HP, Adachi R, Stevens RL. Mast cell-restricted tryptases: structure and function in inflammation and pathogen defense. The Journal of biological chemistry 2007; 282: 20785-9. 219 Dvorak AM, Kissell S. Granule changes of human skin mast cells characteristic of piecemeal degranulation and associated with recovery during wound healing in situ. Journal of leukocyte biology 1991; 49: 197-210. 220 Xue L, Barrow A, Pettipher R. Interaction between prostaglandin D and chemoattractant receptor-homologous molecule expressed on Th2 cells mediates cytokine production by Th2 lymphocytes in response to activated mast cells. Clin Exp Immunol 2009; 156: 126-33. 221 Kanda N, Watanabe S. Histamine enhances the production of nerve growth factor in human keratinocytes. J Invest Dermatol 2003; 121: 570-7. 222 Kohda F, Koga T, Uchi H et al. Histamine-induced IL-6 and IL-8 production are differentially modulated by IFN-gamma and IL-4 in human keratinocytes. J Dermatol Sci 2002; 28: 34-41. 223 Gschwandtner M, Mildner M, Mlitz V et al. Histamine suppresses epidermal keratinocyte differentiation and impairs skin barrier function in a human skin model. Allergy 2013; 68: 37-47. 224 Wahlgren CF, Hagermark O, Bergstrom R. The antipruritic effect of a sedative and a non-sedative antihistamine in atopic dermatitis. The British journal of dermatology 1990; 122: 545-51. 225 Ohsawa Y, Hirasawa N. The antagonism of histamine H1 and H4 receptors ameliorates chronic allergic dermatitis via anti-pruritic and anti-inflammatory effects in NC/Nga mice. Allergy 2012; 67: 1014-22. 226 Schwartz LB. Tryptase: a mast cell serine protease. Methods in enzymology 1994; 244: 88-100. 227 Thakurdas SM, Melicoff E, Sansores-Garcia L et al. The mast cell-restricted tryptase mMCP-6 has a critical immunoprotective role in bacterial infections. The Journal of biological chemistry 2007; 282: 20809-15. 228 Voegeli R, Rawlings AV, Breternitz M et al. Increased stratum corneum serine protease activity in acute eczematous atopic skin. The British journal of dermatology 2009; 161: 70-7. 229 Ribbing C, Engblom C, Lappalainen J et al. Mast cells generated from patients with atopic eczema have enhanced levels of granule mediators and an impaired Dectin-1 expression. Allergy 2011; 66: 110-9. 230 Amon U, Memmel U, Stoll R et al. Comparison of severity scoring of atopic dermatitis values and serum levels of eosinophil cationic protein and mast cell tryptase for routine evaluation of atopic dermatitis. Acta dermato-venereologica 2000; 80: 284-6. 231 Gerdes S, Kurrat W, Mrowietz U. Serum mast cell tryptase is not a useful marker for disease severity in psoriasis or atopic dermatitis. The British journal of dermatology 2009; 160: 736-40.

97

References

232 Tsujii K, Andoh T, Ui H et al. Involvement of Tryptase and Proteinase-Activated Receptor-2 in Spontaneous Itch-Associated Response in Mice With Atopy-like Dermatitis. Journal of pharmacological sciences 2009; 109: 388-95.

98

Appendix

APPENDIX

Figure 31: CD4+ and CD8+ cells in skin lesions of OVA-sensitized mice. A) CD4+ and B) CD8+ T cells were stained and quantified in the dermis of lesional skin of mice. Numbers of

cells per mm2 are shown as the median for each group (n = 5-7). One representative photograph of each group

is shown.

Figure 32: T cell subset cytokine expression in lesional skin. Expression levels of T cell cytokines at the mRNA level in the lesional skin of wildtype and TNF-/- mice were

analyzed by quantitative PCR. The relative expression of A) IL-10, B) IL-4 and INF-γ was measured in

comparison to the housekeeping gene HPRT and is shown as the median for each group (n ≥ 11).

99

Appendix

Figure 33: Proinflammatory cytokine and β-defensins gene expression in lesional skin. Expression levels of cytokines at the mRNA level in the lesional skin of wildtype and TNF-/- mice were analyzed

by quantitative PCR. The relative expression of A) IL-1α, B) IL-1β, C) IL-13, D) IL-33, E) Def B2 and F) Def B3

was measured in comparison to the housekeeping gene HPRT and is shown as the median for each group (n ≥

11).

Figure 34: TSLP and Mast cell levels in mice upon anti TSLP antibody treatment.

A) TSLP mRNA level in wt and TNF-/- mice after intradermal doses of anti-TSLP antibodies B) MCs numbers in

the lesional skin of wt and TNF-/- mice. Median of n ≥4 mice/group.

100

Acknowledgements

ACKNOWLEDGEMENTS

First of all, I would like to express my deepest gratitude to my advisor Prof. Dr. Margitta Worm for giving me the opportunity to do my PhD thesis in her lab at the

Department of Dermatology. Many thanks for her support and supervision and for

her kind advice and help during these years. My special thanks to Dr. Magda Babina for introducing me to the area of

immunomodulation and for her constant help, invaluable suggestions to my work

and during writing publication.

Many thanks to Prof. Dr. Andreas Radbruch for accepting me as PhD student and

for his scientific advice.

I wish to thank Prof. Max Löhning for providing me TNF-/- mice breeding pairs for

my thesis work.

Many thanks to Dana Hoser, Norman Tanner, Barbara Bleher, Tarek Hazzan,

Enya Longmuss and Laura Fleischmann for all their supporting work in my PhD

thesis.

I would like to thanks Dennis Ernst for his great help in the lab and for his

wonderful support during the mouse studies.

I am grateful to all my close friends Dr. Omera Bashir, Dr. Kamalika Mukherjee, Dr. Brinda Selvaraj, Dr. Maria Nassiri and Rajagopal Murgan for helping me in

my hardest situations and for encouraging me. Thank you for your productive

discussions, for proofreading and supporting me with my manuscript(s) and thesis. I

am glad to have you all in my life.

Many thanks to the people from the working group who supported me with

constructive advice, assistance, encouraging words and the wonderful working

atmosphere. Above alI, I would like to express my sincere thanks to all the lab

mates, Juliane Lindner, Dr. Christin Weise, Dr. Kiran Kumar, Dr. Gennadiy Drozdenko, Dr. Sabine Dölle, Kristina Heins, Marcel Wittenberg, Sandra Treptow, Davender , Dr. Guido Heine, Dr. Bernhard Ay for their cooperation and

support during my stay. I also thank previous lab colleagues, Dr. Björn Hartmann

and Dr. Kerstin Geldmeyer-Hilt for their valuable suggestions.

101

Acknowledgements

A special thanks goes to Sven Guhl and Metin Artuc for giving me useful technical

and scientific advice during the whole course of my PhD thesis, especially for

human keratinocytes and mast cell culturing.

I would like to thank and treasure the good times I spent with all my friends in Berlin.

Thank you for making my stay in Berlin a pleasant one. Lastly, I would like to show gratitude to my family for all their support and their belief

in me throughout my life. I feel myself lucky to have you all. Finally, I would like to

thank my husband and partner Krishna Kumar for his unconditional love, patience

and support during this period of time.

102

Declaration

SELBSTÄNDIGKEITSERKLÄRUNG / DECLARATION

Hiermit versichere ich, Vandana Kumari, die vorliegende Dissertation selbständig

erarbeitet und verfasst zu haben. Es wurden keine weiteren Quellen und Hilfsmittel

als die hier angegebenen verwendet.

I hereby declare that I, Vandana Kumari, have worked and wrote this dissertation

independently and did not use other than the listed support. This thesis does not

exist neither in the same or similar form nor is it submitted to another examination

procedure.

103

mechanisms underlying the regulatory function of tumor

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