rheumatoid arthritis, psoriatic arthritis and psoriasis

252 Pages·2011·3.3 MB·English

Hueber, Axel Johannes (2011) The role of the cytokines IL-17A and IL-33
in inflammatory arthritis and psoriasis.
PhD thesis.

http://theses.gla.ac.uk/2629/

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Glasgow Theses Service
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The role of the cytokines IL-17A and IL-33
in inflammatory arthritis and psoriasis
by
Axel Johannes Hueber
MD

Submitted in fulfillment of the requirements for the degree of Doctor of
Philosophy

Division of Immunology, Infection & Inflammation
Faculty of Medicine
University of Glasgow
2
Abstract
The inflammatory autoimmune diseases rheumatoid arthritis, psoriatic arthritis
and psoriasis have seen a break through in therapy by targeting cytokines in the
last decade. Interleukin-17A, a potential new target, is considered as a crucial
player in rheumatoid arthritis, and has been suggested to be produced by CD4+ T
cells (Th17 cells). I explored the cellular sources of IL-17A in human established
RA synovium. Surprisingly, only a small proportion of IL-17 positive cells were T
cells without expression of a Th17 marker CCR6. Unexpectedly, the majority of
IL-17A expression colocalized within mast cells. These data do not contradict a
crucial role for IL-17A in RA pathogenesis, however, suggest that in addition to
Th17 cells, cells of the innate immune system, particularly mast cells, may be an
important component of the effector IL-17A response.
Psoriasis is a common chronic autoimmune disease of the skin characterized by
hyperplasia of epidermal keratinocytes with associated inflammation. IL-33 is a
new member of the IL-1 superfamily that signals through the ST2 receptor and
was originally defined as an inducer of T helper 2 (Th2) cytokines. Recently
broader immune potential has been discovered for IL-33 particularly via mast
cell activation. With its expression at body barrier surfaces it is assumed to act
as an alarmin. In this thesis I demonstrate that IL-33 expression is up-regulated
in the epidermis of psoriatic lesions, compared to healthy skin, thus indicating
that IL-33 may be a mediator regulating crosstalk between keratinocytes and
infiltrating immune cells in psoriatic plaques. In a phorbol ester-induced model
of skin inflammation ST2-/- mice exhibited reduced cutaneous inflammatory
responses compared to WT mice. Furthermore, consecutive injections of IL-33
into the ears of mice induced a psoriasis-like inflammatory lesion. This was
partially mast cell dependent and cellular analysis demonstrated recruitment of
neutrophils to the ear. This concludes that IL-33, via activation of mast cells and
recruitment of neutrophils, may play a role in psoriasis plaque inflammation.
In the last part of the thesis I tested if nanoparticles can be utilized to image
cytokine driven inflammation. Bio-linkages with protein-nanoparticles have been
established and in vivo detection of nanoparticles performed. This final
interdisciplinary outlook demonstrates a still to be established/finalized method
with great potential.
3
Table of Contents
Abstract ..................................................................................... 2
Table of Contents.......................................................................... 3
List of Tables............................................................................... 6
List of Figures.............................................................................. 7
List of Figures.............................................................................. 7
Acknowledgement......................................................................... 9
Author's declaration .....................................................................10
Abbreviations..............................................................................11
1 Introduction .......................................................................15
1.1 Autoimmune inflammation – rheumatoid arthritis, psoriatic arthritis and
psoriasis..................................................................................17
1.1.1 The spectrum of clinical presentation of inflammatory arthropathies
and psoriasis............................................................................17
1.1.2 Pathogenesis of rheumatoid arthritis.....................................28
1.1.3 Pathogenesis of psoriatic arthritis and psoriasis ........................31
1.1.4 Pathogenic immune cells in inflammatory arthritis.....................32
1.1.5 Cytokines in synovitis and dermatitis.....................................34
1.1.6 Mouse models of inflammatory skin disease.............................40
1.2 Th17 cells and their role in rheumatoid arthritis/psoriasis ..............42
1.3 The role of the cytokine Interleukin 33 in health and disease...........51
1.3.1 IL-33 in the context of its IL-1 receptor / Toll like receptor family
members ................................................................................51
1.3.2 Structure of IL-33............................................................56
1.3.3 Tissue localisation and cellular expression of IL-33.....................61
1.3.4 Release of IL-33..............................................................61
1.3.5 IL-33 receptor signalling via ST2 and IL1RacP ...........................63
1.3.6 Cellular biological functions of IL-33......................................67
1.3.7 Regulation of IL-33...........................................................68
1.3.8 IL-33 in disease...............................................................68
1.4 Nanoparticles in inflammation................................................72
1.4.1 What are nanoparticles? ....................................................73
1.4.2 Magnetofluorescent nanoparticles ........................................76
1.4.3 Fluorescent nanoparticles..................................................80
1.4.4 Surface-enhanced Raman spectroscopy (SERS)..........................81
1.5 Aims of this thesis..............................................................82
2 Material and Methods............................................................83
2.1 General reagents & buffers ...................................................83
2.1.1 Materials and reagents......................................................83
2.1.2 Buffers and culture media..................................................83
2.2 Patients ..........................................................................83
2.3 Skin biopsy.......................................................................84
2.4 Tissue preparation..............................................................84
2.4.1 Paraffin embedded tissue...................................................85
2.4.2 Frozen tissue..................................................................85
2.5 ImmunHistoChemistry (IHC) of paraffin embedded sections.............85
2.5.1 Single staining for light microscopy.......................................85
2.5.2 Double staining for light microscopy......................................87
2.5.3 Double staining for fluorescent microscopy..............................87
2.5.4 Quantification of fluorescent IHC .........................................89
4
2.5.5 Mast cell staining using Toluidine blue ...................................89
2.6 ELISA..............................................................................89
2.7 Luminex cytokine analysis.....................................................89
2.8 Cell culture......................................................................89
2.8.1 Culture of adherent cells...................................................89
2.8.2 Culture of suspension cells.................................................90
2.8.3 Purification of monocytes ..................................................90
2.8.4 Splenocyte harvest...........................................................91
2.9 FACS analysis....................................................................91
2.10 Animals...........................................................................92
2.11 IL-33 related mouse experiments ............................................94
2.11.1 TPA skin inflammation model ...........................................94
2.11.2 Cytokine ear injection model............................................96
2.11.3 Wound healing biopsy model............................................98
2.11.4 Analysis of mouse embryos .............................................101
2.12 Production of a K14-IL-33 construct ........................................101
2.12.1 Cloning of K14-IL-33 .....................................................101
2.12.2 Transfection of K14-IL-33 in HaCaT ...................................102
2.13 Nanoparticle methods.........................................................103
2.13.1 Protein linked SERRS active nanoparticles ...........................103
2.13.2 Western blot of protein-linked NP.....................................104
2.13.3 ETA-NP binding capacity ................................................105
2.13.4 HeLa cell activation with TNF-α and blockade with ETA-NP.......106
2.13.5 Nanoparticles in vivo ....................................................106
2.13.6 Carrageenan foot paw injection model...............................108
2.14 Statistical analysis.............................................................108
3 Interleukin-23 and Interleukin-17 in inflammatory arthropathies ...109
3.1 Aim and Introduction..........................................................110
3.2 IL-23 expression in inflammatory arthropathies ..........................112
3.2.1 Synovial expression of IL-23p19...........................................112
3.2.2 Expression of IL-23 in synovial fluid......................................113
3.3 The source of IL-17 in rheumatoid arthritis................................116
3.3.1 IL-17 is expressed in RA synovium but only scarce in OA synovium .116
3.3.2 IL-17 is rarely expressed by Th17 cells in RA synovium ...............118
3.3.3 IL-17 is produced by innate immune cells mainly mast cells.........126
3.4 Discussion & conclusion.......................................................129
4 Analysis of Interleukin-33 as an alarmin in psoriasis....................133
4.1 Aim and Introduction..........................................................134
4.2 IL-33 and ST2 is expressed in inflammatory tissue such as psoriasis...136
4.2.1 Expression of IL-33 and ST2 in autoimmune diseases..................136
4.2.2 Interaction of IL-33 with mast cells......................................150
4.3 The role of IL-33 in skin inflammation mouse models....................152
4.3.1 TPA induced skin inflammation...........................................152
4.3.2 IL-33 intradermal ear injections – a model of skin inflammation....157
4.4 Addendum - Miscellaneous biologic questions surrounding IL-33.......169
4.4.1 IL-33 in wound healing.....................................................170
4.4.2 IL-33 expressed under the K14 promotor................................172
4.4.3 Embryonic function of IL-33...............................................175
4.5 Discussion.......................................................................178
5 The use of nanoparticles to image inflammation ........................184
5.1 Aim and Introduction..........................................................185
5.2 Size and form of nanoparticles..............................................187
5.3 Linking proteins to nanoparticles ...........................................189
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5.3.1 Testing quantities of ETA on linked nanoparticles.....................191
5.3.2 Biological binding properties of ETA linked nanoparticles to TNF-α 193
5.3.3 Biological function of ETA linked nanoparticles in HeLa cells........197
5.4 The detection of nanoparticles in vivo.....................................201
5.4.1 SERRS profile of NP (Strathclyde) ........................................201
5.4.2 Detection of NPs in tissue .................................................203
5.4.3 The detection of NPs in inflammation...................................205
5.4.4 Switch of detection laser and Nanoparticles to Nanotags ............209
5.4.5 SERRS profile of nanotags (NT) ...........................................209
5.4.6 Detection of i.v. administered NT440 in vivo...........................211
5.4.7 Ex vivo SERRS mapping analysis of spleen...............................213
5.4.8 Multiplexing of NT in vivo and ex vivo...................................215
5.5 Conclusion and Discussion....................................................217
6 Discussion & conclusion.......................................................220
7 References.......................................................................224
8 Publications......................................................................251
6
List of Tables
Table 1.1 criteria 1987 Criteria for the classification of rheumatoid arthritis (1)
...............................................................................................19
Table 1.2 The 2010 American College of Rheumatology/European League Against
Rheumatism classification criteria for rheumatoid arthritis (adapted from (2)).19
Table 1.3 CASPAR criteria for classification of PsA (adapted from (8))...........20
Table 1.4 Comparison of rheumatoid arthritis, psoriatic arthritis, ankylosing
spondylitis and osteoarthritis............................................................24
Table 1.5 Susceptibility genes in arthritis.............................................29
Table 1.6 Selected cytokines implicated in pathogenesis of RA, PsA and psoriasis
...............................................................................................38
Table 1.7 IL-1 family – members and nomenclature .................................53
Table 2.1 Antibodies for IHC.............................................................88
Table 2.2 ELISA cytokines and companies .............................................88
Table 2.3 Mouse strains used in experiments .........................................93
Table 3.1 Percentage of double positive cells compared to IL-17+ cells.........128
7
List of Figures
Figure 1.1 Cytokine receptor interaction..............................................27
Figure 1.2 Picture of synovial joint inflammation....................................30
Figure 1.3 T helper cell differentiation (overview). .................................43
Figure 1.4 Th17 cell differentiation in mice...........................................46
Figure 1.5 Th17 polarisation in humans................................................46
Figure 1.6 IL-18 receptor complex......................................................55
Figure 1.7 Structure of IL-33 HTH like motif..........................................58
Figure 1.8 IL-33 structural components and cleavage sites.........................60
Figure 1.9 IL-33 binding to ST2 and IL1RacP signals via MyD88.....................66
Figure 1.10 Nanoparticle – size comparison...........................................74
Figure 2.1 Overview of the timeline - TPA skin inflammation model..............95
Figure 2.2 IL-33 intradermal ear injection.............................................97
Figure 2.3 Wound healing model.......................................................100
Figure 2.4 SERRS imaging ex vivo. .....................................................107
Figure 3.1 Synovial expression of IL-23 in inflammatory arthropathies. .........114
Figure 3.2 Detection of IL-23 in synovial fluid. ......................................115
Figure 3.3 IL-17A expression in RA/OA synovium....................................117
Figure 3.4 Establishing staining for CCR6 in RA......................................119
Figure 3.5 Doublestaining IL-17 and CCR6 by light IHC .............................120
Figure 3.6 Back to back staining CD3/CCR6 and IL-17..............................122
Figure 3.7 Doublestaining of Th17 cell markers and IL-17A........................124
Figure 3.8 Doublestaining of Th17 marker CCR6 with IL-17A. .....................125
Figure 3.9 Doublestaining of IL-17 pos. cells with CD68............................127
Figure 3.10 Mast cells express IL-17A in RA synovium. .............................127
Figure 3.11 RORC dependent IL-17A production by CD34+ derived mast cells..132
Figure 4.1 Hypothesis for the role of IL-33 in psoriasis.............................135
Figure 4.2 IL-33 in tonsil – work up of IHC staining..................................137
Figure 4.3 IL-33 expression in healthy skin...........................................137
Figure 4.4 IL-33 in psoriasis.............................................................138
Figure 4.5 IL-33 is not expressed by basal keratinocyte stem cells...............140
Figure 4.6 ST2 expression in healthy skin.............................................142
Figure 4.7 ST2 expression in psoriatic skin. ..........................................143
Figure 4.8 IL-33 expression in rheumatoid arthritis.................................145
Figure 4.9 ST2 and IL-33 expression in rheumatoid arthritis.......................146
Figure 4.10 IL-33 expression in synovial fibroblasts.................................147
Figure 4.11 sST2 expression in PsA and healthy control serum and PsA and OA
synovial fluid..............................................................................149
Figure 4.12 Expression of ST2 in HMC-1 and HaCaTs................................151
Figure 4.13 Stimulation of HMC-1 with IL-33.........................................151
Figure 4.14 TPA induced skin inflammation – Role of IL-33/ST2 ..................153
Figure 4.15 TPA induced skin inflammation – Ki67 proliferation analysis........154
Figure 4.16 TPA induced skin inflammation ST2 dependency resolved after 3
days.........................................................................................156
Figure 4.17 TPA induced skin inflammation in mast cell deficient mice – no
difference after 3 days. .................................................................156
Figure 4.18 Validation of the biological function of recombinant IL-33..........158
Figure 4.19 Model of injection and in vivo biological activity of IL-33...........158
Figure 4.20 IL-33 induces ear swelling in an ear injection model.................160
Figure 4.21 IL-33 injection- histological readout (H&E)............................160
8
Figure 4.22 IL-33 ear injection –infiltration of mast cells and eosinophils.......162
Figure 4.23 IL-33 ear inflammation is ST2 dependent ..............................164
Figure 4.24 IL-33 initial effect in ear inflammation is partial dependent on mast
cells ........................................................................................166
Figure 4.25 Cellular recruitment by IL-33 ............................................168
Figure 4.26 ST2 deficiency does not influence wound healing ....................171
Figure 4.27 Construct and design of the K14 IL-33 plasmid........................173
Figure 4.28 Transfection of HaCaTs with K14 IL-33 .................................174
Figure 4.29 Embryo preparation day D9.5............................................176
Figure 4.30 IL-33 is not expressed in the dorsal aorta day 9.5 or 10.5...........177
Figure 4.31 Splice variants of IL-33....................................................183
Figure 5.1 SEM on nanoparticles (nanotags)..........................................188
Figure 5.2 Schematic linking of proteins to NPs. ....................................190
Figure 5.3 Total protein on ETA linked nanoparticles ..............................192
Figure 5.4 Competition ELISA to test TNF-α binding to NPs........................195
Figure 5.5 Biological binding properties of ETA-NPs. ...............................195
Figure 5.6 Biological binding properties of ETA-NT440.............................196
Figure 5.7 Establishing TNF stimulation of Hela cells with ETA blockade........198
Figure 5.8 IL-6 production by TNF stimulated HeLa cells blocked with Au-ETA.198
Figure 5.9 Nanoparticles aggregate over time.......................................200
Figure 5.10 Linkage and bioactivity of ETA-NP (Au) with BSA. ....................200
Figure 5.11 SERRS profile of NIR-Au-NP...............................................202
Figure 5.12 Detection of Nanoparticles in tissue ....................................204
Figure 5.13 Detection of NPs in the carragean footpad injection model.........206
Figure 5.14 Tracking of Nanoparticles to the site of inflammation...............208
Figure 5.15 SERRS profile of NT440 and NT420 ......................................210
Figure 5.16 SERRS signal of NT440 in tissue..........................................212
Figure 5.17 SERRS image of a spleen section.........................................214
Figure 5.18 SERRS signals of liver and spleen in NT injected mice. ..............216
9
Acknowledgement
I would like to thank Professor Iain McInnes for his great support during the 3 ½
years in Glasgow. Professor McInnes has been an inspiring clinical and scientific
mentor and has always tried to help developing my career in the best possible
ways throughout this period and beyond.
This thesis would not have been possible without the help and support from my
laboratory and clinical colleagues. I am particularly grateful to Dr Ashley Miller
and Dr Darren Asquith for their guidance throughout these years. Day to day
technical and scientific supervision with a strong support of activities made the
time flying by. This was great supported by Lucy Ballantine.
In the CRD lab group I would also like to express my gratitude to Jim “IHC
warlock” Reilly and Shauna Kerr (“IHC witch”). Taking myself as the worst
apprentice on board to teach what real “bucket” science is like, was very kind
and I appreciate the knowledge and insights I have learnt. Also I want to thank
Ashley Gilmour and Alastair Fraser for all their help.
I would like to thank Mr Neal Millar who gave me the opportunity to see how real
doctors work and for a successful orthopaedic/rheumatology collaboration.
I would also like to thank Professor Sturrock, Dr Max Field, Dr David McCarey and
especially Dr Hilary Wilson but also the nurses for providing me with patient
samples. In addition I would like to thank the patients from the rheumatology
clinic who donated samples for this project.
Thanks to Dr Derek Gilchrist for helping me with the IL-33 biology work and Dr
Ross Stevenson for insights and collaboration regarding nanoparticle science.
Special thanks also to my wife and family (Ralf for 3 Glasgow visits including for
the surprise visit while I was taking a shower…). My wife, especially, has
sacrificed a lot to allow me to have an enjoyable life and successful career and
for that I will always be extremely thankful.
The German Research Foundation (DFG) funded three years of this project.