The IL-1 family in relation to psoriasis Rosella Dorothy Amy Doble Submitted in accordance with ...

274 Pages·2014·7.28 MB·English

The IL-1 family in relation to psoriasis
Rosella Dorothy Amy Doble
Submitted in accordance with the requirements for
the degree of Doctor of Philosophy
The University of Leeds
Faculty of Biological Sciences
July 2014
The candidate confirms that the work submitted is his/her own, except
where work which has formed part of jointly authored publications has
been included. The contribution of the candidate and the other authors to
this work has been explicitly indicated below. The candidate confirms
that appropriate credit has been given within the thesis where reference
has been made to the work of others.
Chapter 3 – based on Wittmann, M., Doble R., Bachmann M.,
Pfeilschifter J., Werfel T., Mühl H., IL-27 Regulates IL-18 binding
protein in skin resident cells. PLoS One, 2012. 7(6): p. e38751.
Conceived and designed the experiments: MW HM RD. Performed the
experiments: RD MB. Analysed the data: RD MW HM MB. Contributed
reagents/materials/analysis tools: JP TW HM MW. Wrote the paper: RD
MW HM TW.
Chapter 4 – based on Doble, R., McDermott MF., Cesur O., Stonehouse
NJ., Wittmann M., IL-17A RNA aptamer: possible therapeutic potential
in some cells, more than we bargained for in others? J Invest Dermatol,
2014. 134(3): p. 852-5. Conceived and designed experiments: RD, NS,
MW. Performed the experiments: RD, OC. Analysed the data: RD.
Contributed reagents/materials/analysis tools: MW NS MD. Wrote the
paper: RD, MW, NS.
This copy has been supplied on the understanding that it is copyright
material and that no quotation from the thesis may be published without
proper acknowledgement.
© 2014 The University of Leeds and Rosella Dorothy Amy Doble
i
Acknowledgements
I would firstly like to say a massive thank you to my supervisors, Dr Miriam
WittmannandDrMartin Staceyfor theirhugeamounts ofsupport and guidanceover
the last 4 years, their input has allowed me to establish a wide range of techniques
with confidence. I would also like to thank Professor Dennis McGonagle and Dr
Neil Turner for their critique and input of ideas during the last 4 years. I am also
hugely grateful to Professor Nicola Stonehouse and Dr Andrew Macdonald for their
collaborative projects for which their guidance has been invaluable. I would also like
to thank Chris Wasson for his patience and helpful guidance in construction of skin
equivalents, as well as Özlem Cesur and Sophie Forrest for their help with my
aptamer work. I would like to thank members of the Wittmann and Stacey group,
Ade Alase and Tom Macleod for making the lab a pleasant place to work. I would
also like to thank everyone who has been in the lab over the last 4 years especially
those who have kept me sane and made the last 4 years enjoyable. I would like to
thank Laura Knight, Hannah Kyle, Chris Wasson, Emma Prescott, James Findlay
and Laura White for the regular tea/gossip breaks and who have all during the 4
years kept my spirits up. I am very grateful for all the support from my friends and
familywho despite not knowing what I am talking about still share in the excitement
and frustration of research. I would lastly like to thank Mark for his unwavering
support in listening and understanding the ups and downs of doing a PhD, no matter
howgrumpyIwas.
ii
Abstract
Psoriasis is a common chronic inflammatory skin disease which can also affect the
joints. Its pathogenesis is still to be fully elucidated and involves a wide range of
inflammatory mediators, tissue and immune cells. At present, there is no treatment
available to cure psoriasis. Although biologics have considerably improved the
treatment of the most severe cases there is still a pressing clinical need to improve
therapy for specific disease subtypes (e.g. pustular psoriasis) and the vast majorityof
patients suffering from psoriasis classified as mild to moderate. In particular,
efficient andwell toleratedtopical approaches are lacking.
This work has focused 1) on advancing our understanding of IL-36 cytokines which
are recognised for their significance in pustular psoriasis, 2) on identifying
endogenous disease limiting mediators such as IL-18 binding protein and how they
could be manipulated in a therapeutic approach and 3) on IL-17 neutralising RNA
aptamers as tools fortopical therapy.
Main results include the identification of biologic activity of processed and non-
processed IL-36 members. N-terminal cleavage is required to increase activity of all
IL-36 members. The protease responsible for IL-36RA processing was elucidated.
Neutrophil proteases as well as kallikrein 7 can cleave pro-inflammatory IL-36
members. However, a second processing step seems necessaryfor full activation and
thepotentiallyresponsibleaminopeptidase remains tobeidentified. Secondly,it was
found that human primary fibroblasts produce significant levels of IL-18BP, which
controls pro-inflammatory function of IL-18. Endogenous IL-18BP can be induced
by IL-27 which, when given in combination with hydrocortisone does not induce
pro-inflammatory responses. Thirdly, an IL-17 specific aptamer was verified to
block IL-17A activity in fibroblast and fibroblast Th17 co-cultures but not in
keratinocyte cultures. Significant uptake of the RNA aptamer by keratinocytes was
identifiedas potentiallyresponsibleforthelackof neutralisingcapacity.
iii
Table of Contents
Acknowledgements......................................................................................................ii
Abstract......................................................................................................................iii
List of Figures.............................................................................................................ix
List ofTables............................................................................................................xiii
Abbreviations............................................................................................................xiv
Chapter1- Introduction...............................................................................................1
1.1 Introduction........................................................................................................2
1.2Anatomyoftheskin...........................................................................................3
1.3Skininflammation..............................................................................................5
1.3.1 Initiationofskininflammation.......................................................................................6
1.3.2Maintenance andresolutionofskininflammation.......................................................11
1.3.3Chronicinflammation...................................................................................................13
1.4Psoriasis andpsoriaticarthritis.........................................................................14
1.4.1Clinical characterisationofpsoriasis............................................................................15
1.4.2Pathogenesis ofpsoriasis..............................................................................................17
1.4.3Associatedco-morbidities inpsoriasis.........................................................................20
1.4.4Geneticimplications inpsoriasis..................................................................................21
1.5The IL-1family................................................................................................23
1.5.1Activityandprocessingofthe IL-1familymembers...................................................26
1.5.2The IL-1 receptors andnegativeregulators..................................................................30
1.5.3 IL-36,asubgroupofthe IL-1family............................................................................33
1.5.4 Interleukin-18................................................................................................................38
1.6 Interleukin-27...................................................................................................41
1.6.1 IL-27receptor and signalling........................................................................................42
iv
1.6.2Pro-inflammatoryroles of IL-27...................................................................................43
1.6.3Anti-inflammatoryroles of IL-27.................................................................................44
1.7Proteolyticcleavage ofinflammatorymediators in theskin............................45
1.7.1Proteases intheskin......................................................................................................45
1.7.2 Inactivationofinflammatorymediators byproteolyticcleavage.................................50
1.7.3Activationofinflammatorymediators byproteolyticcleavage...................................50
1.8RNAaptamers..................................................................................................52
1.8.1ProductionofRNAaptamers........................................................................................52
1.8.2Potential clinical useofRNAaptamers........................................................................55
1.8.3Potential mechanisms ofinternalisationofoligonucleotides.......................................56
1.8.4Thechallenges ofusingRNAaptamers fortherapeutics.............................................57
1.9 Interleukin-17...................................................................................................58
1.9.1 IL-17receptors and signallingpathways......................................................................58
1.9.2Functional roles of IL-17..............................................................................................60
1.9.3Theroleof IL-17indisease..........................................................................................61
1.10Project aims....................................................................................................62
Chapter2–Materials andmethods............................................................................64
2.1Materials...........................................................................................................65
2.1.1Buffers used..................................................................................................................66
2.1.2Cell lines used...............................................................................................................67
2.2General methods...............................................................................................67
2.2.1Obtainingprimarycells................................................................................................68
2.2.2Cultureofprimarycells andcell lines..........................................................................68
2.2.3 Outgrowth of fibroblasts from healthy and patient derived skin and tendon
biopsies..................................................................................................................................69
2.2.4Transfectionofprimarycells and cell lines...................................................................69
v
2.2.5Peripheral bloodcollection...........................................................................................69
2.2.6 IsolationofPBMCs from wholeblood.........................................................................70
2.2.7CD4+Tcell isolationfrom PBMCs –usingMACS®cell separation.........................70
2.2.8CultureofprimaryCD4+Tcells..................................................................................70
2.2.9Enrichment ofCCR6+Tcells from CD4+ Tcells.......................................................71
2.2.10 IsolationofPMNs from wholeblood.........................................................................71
2.2.11Constructionofskinequivalents.................................................................................72
2.2.12Enzyme-linkedimmunosorbent assay(ELISA).........................................................72
2.2.13Flourescenceassistedcell sorting(FACS).................................................................73
2.2.14Quantitativereal-timepolymerasechainreaction(qRT-PCR)...................................73
2.2.15Fixingandstainingcells forfluorescenceimaging....................................................73
2.2.16 Di-AminoBenzidine (DAB) staining of paraffin embedded skin
equivalents.............................................................................................................................74
2.3Molecularbiology............................................................................................75
2.3.1SDS-Polyacrylamidegel electrophoresis (PAGE).......................................................75
2.3.2Westernblotting............................................................................................................75
2.3.3Agarose gel electrophoresis..........................................................................................76
2.3.4Conventional PCRforcloning......................................................................................76
2.3.5Restrictiondigest..........................................................................................................76
2.3.6 Ligation.........................................................................................................................77
2.3.7Transformation..............................................................................................................77
2.3.8Expressionof IL-1 familymembers in E.coli..............................................................77
2.3.9Purificationofproteinusingsizeexclusioncolumn.....................................................78
2.3.10RemovingSUMO from relevant constructs...............................................................79
2.3.11RNAaptamer chemical synthesis...............................................................................79
Chapter3– IL-18bindingproteinis up-regulated byIL-27.....................................80
vi
3.1 Introduction......................................................................................................81
3.2 IL-18BP expressionfollowingIL-27stimulation............................................83
3.3Timedependent expressionof IL-18BP andCXCL10inresponseto IL-27...87
3.4 IL-27activates STAT1downstream ofits receptor.........................................90
3.5 Understanding the different expression levels of IL-18BP and CXCL10 at
different timepoints...............................................................................................93
3.6Discussionandfuture work..............................................................................96
Chapter 4 – Establishing the efficacy of the anti-IL-17A RNA aptamer, Apt21-2 in
skinresident cells.....................................................................................................101
4.1 Introduction....................................................................................................102
4.2StructureofApt 21-2......................................................................................104
4.3EfficacyofApt21-2inhumanprimaryfibroblasts........................................105
4.4EfficacyofApt21-2inhumanprimarykeratinocytes....................................109
4.5UnexpecteduptakeofApt21-2.......................................................................110
4.6 Immune activationbyApt21-2.......................................................................116
4.7Discussionandfuture work............................................................................119
Chapter5–Elucidatingtheroleof IL-36cleavage.................................................126
5.1 Introduction....................................................................................................127
5.2Responseofprimaryskinresident cells to IL-36...........................................128
5.2.1 IL-36α ......................................................................................................................... 129
5.2.2 IL-36β ......................................................................................................................... 131
5.2.3 IL-36γ.......................................................................................................................... 133
5.3.4Effects of IL-36on PMNandTcell activation..........................................................135
5.3Cleavageof full length IL-36.........................................................................135
5.4Productionofrecombinant tagged IL-36.......................................................136
5.5 Identifyingputativeproteases responsiblefor IL-36 cleavage......................140
vii
5.6 IL-36cleavageproducts identified.................................................................145
5.7Productionofidentifiedcleavedproducts......................................................149
5.8Responseofhumanprimaryfibroblasts tothe cleaved IL-36proteins..........150
5.9Responseofskinequivalent models tostimulationbyIL-36........................156
5.10 IL-38as apossible IL-36 antagonist inhumanfibroblasts..........................161
5.11Mammalianexpressionof IL-36..................................................................162
5.12Discussionandfuturework..........................................................................164
Chapter6–Discussion............................................................................................175
6.1Thebalancebetween pro-andanti-inflammatorymediators.........................176
6.2Control ofpro-inflammatorymediators byproteases....................................177
6.3Possiblenovel therapeutics inpsoriasis.........................................................179
6.4Conclusion......................................................................................................181
References................................................................................................................183
Appendix..................................................................................................................219
viii
List of Figures
Figure1-1. Diagrammaticrepresentationofthelayers oftheskin..............................5
Figure1-2. Diagrammaticrepresentationofinitiationofskininflammation...........10
Figure1-3.Clinical characteristics ofthepsoriaticsubtypes....................................15
Figure1-4. Diagrammaticrepresentationofpsoriaticinflammation.........................20
Figure1-5. Adiagrammaticrepresentationofthe IL-1 receptorfamily....................31
Figure 1-6. Diagrammatic representation of the IL-12 family of cytokines and
receptors.....................................................................................................................42
Figure1-7.SummaryoftheSELEXprocess.............................................................54
Figure 1-8. Diagrammatic representation of the IL-17 receptor subunits and the
pairings formedtoproducefunctional receptors.......................................................59
Figure 3 - 1. IL-27 dose dependently up-regulates IL-18 binding protein (BP) in
humankeratinocytes...................................................................................................85
Figure 3 – 2. IL-27 dose dependentlyincreases levels of IL-18BP in human primary
fibroblasts...................................................................................................................87
Figure 3 – 3. mRNA levels of IL-18BP are up-regulated in IL-27 stimulated human
primaryfibroblasts.....................................................................................................88
Figure 3 – 4. IL-18BP is released in a time dependent manner in response to IL-27.
....................................................................................................................................89
Figure 3 – 5. CXCL10 is differentially expressed compared to IL-18BP in response
to IL-27 or IFNγ. ........................................................................................................ 90
Figure 3 – 6. IL-27 induces IL-18BP via a STAT1 pathway and the proximal GAS
siteinthe IL-18BP promoteris crucial for geneactivation.......................................92
Figure 3 – 7. IL-18BP RNA does not show increased stability and constant IL-27
receptoractivationis not requiredforprolongedproductionof IL-18BP.................94
Figure3–8.CXL10,but not IL-18BP releaseis preventedbyhydrocortisone........95
Figure4–1. IL-17Aaptamer-Apt21-2..................................................................105
ix