Discrimination between CMV infection and Alzheimer's disease as

133 Pages·2014·3.31 MB·English

Discrimination between CMV infection and
Alzheimer's disease as driving forces for immune
senescence

Dissertation
der Mathematisch-Naturwissenschaftlichen Fakultät
der Eberhard Karls Universität Tübingen
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
(Dr. rer. nat.)

vorgelegt von
David Goldeck
aus Fulda

Tübingen
2014
Tag der mündlichen Qualifikation: 19.05.2014
Dekan: Prof. Dr. Wolfgang Rosenstiel
1. Berichterstatter: Prof. Dr. Graham Pawelec
2. Berichterstatter: Prof. Dr. Hans-Georg Rammensee
Acknowledgements
This thesis wouldn’t have been possible without many helping hands and minds which I grate-
fullythank:
• Prof. Dr. GrahamPawelec
fortheopportunitytoworkintheTATIgroupandforguidingandsupervisingme,correc-
tingandimprovingmytalks,postersandpresentations;
• Prof. Dr. Hans-GeorgRammensee
forthegreattimeattheGRK794andforactingasmysecondreviewer;
• Dr. AnisLarbi
forbeingmysupervisor,whotaughtmealot,andcameupwiththeinterestingtopic;
• the helpful colleagues which worked hard in the lab to perform the studies presented in
thisthesis,namely
IftikharAlam,MarkusClaus,LillyOettinger,MariavaleriaPellicanó,SimonWalker;
• all other TATI members for teaching me methods, for fruitful discussions and for a warm
andjoyfulatmospherewheresciencebringshappinessinyourlife,
Wim Adriaensen, Mathias Blaurock, Silvio Buffa, Evelyna Derhovanessian, Svetlana Di
Benedetto, Andrea Groeger, Lutz Grossmann, Karin Hähnel, Florian Heubach, Kyriaki
Ioannou,NicoleJanssen,JithendraKiniBailur,AlexanderMartens,ArnikaRehbein,Flavia
Ribeiro, Graziella Rubino, Chris Shipp, Lisa Speigl, Christina Stutz, Kilian Wistuba-
Hamprecht,HenningZelba;
• WolfgangKunert
forgreatadditionalITsupport;
• allcollaboratingpartnerswhoprovidedsamplesandgoodadvice
Prof. Klaus Hamprecht, Prof. Dorothee Wernet, Prof. Calogero Caruso, Prof. Guiseppina
Colonna Romano, Prof. Inga Zerr, Dr. Christian Schmidt, Prof. Tamàs Fülöp , Prof.
RobertoPaganelli;
• my family, especially my parents and siblings, for supporting me to come that far and
helpingoutwithmythesis;
• andallwhichIhaveforgotten.
I
Contents
1 Introduction 1
1.1 Typesofdementiaandtheirdiagnosis . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.1 Mildcognitiveimpairment(MCI)andthedifficultiesofitsdiagnosis . . 3
1.1.2 Alzheimer’sDisease(AD) . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.1.3 Vasculardementia(VaD) . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.1.4 Differentialdiagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.1.5 TheFolsteinTestandtheMMSEvalue . . . . . . . . . . . . . . . . . . 11
1.1.6 TreatmentsforAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.2 ImmunesignaturesofhealthyindividualsandADpatients . . . . . . . . . . . . 13
1.2.1 T-cellheterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.2.2 T-cellphenotypesinhealthyelderlyandADpatients . . . . . . . . . . . 16
1.2.3 Tregphenotypesandtheirassociationwithageinganddementia . . . . . 17
1.2.4 B-cellphenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.2.5 SystemicinflammationinAD . . . . . . . . . . . . . . . . . . . . . . . 19
1.2.6 PlasmaandserumcytokinelevelsinthecontextofinflammationinAD . 20
1.3 CMV,dementiaandtheimmunesystem . . . . . . . . . . . . . . . . . . . . . . 21
1.3.1 AssociationbetweenCMVinfectionandT-celldifferentiation . . . . . . 21
1.3.2 HerpesvirusesandAD . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
1.4 BraininfiltrationbyperipheralimmunecellsalongachemokinegradientinAD . 23
1.5 Rationaleofthepresentstudy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2 MaterialsandMethods 28
2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.1.1 Buffersandreagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.1.2 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.1.3 Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.1.4 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.2 Samplecollectionandprocessing . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.2.1 Collaboratorswhoprovidedsamples . . . . . . . . . . . . . . . . . . . . 31
2.2.2 PBMCisolation,freezingandstorage . . . . . . . . . . . . . . . . . . . 32
2.2.3 Frozenwholebloodstorage . . . . . . . . . . . . . . . . . . . . . . . . 32
2.2.4 Preparationofsamplesforflowcytometricanalysis . . . . . . . . . . . . 32
2.2.5 Experimentaldesign . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
II
2.2.6 StainingcellsurfaceproteinsandintracellularFoxp3withfluorochrome-
conjugatedantibodiesforflowcytometricanalysis . . . . . . . . . . . . 34
2.2.7 Intracellularcytokinestaining . . . . . . . . . . . . . . . . . . . . . . . 35
2.2.8 TestingPBMCuptakeofamyloidbeta . . . . . . . . . . . . . . . . . . . 35
2.3 TestingCMVserostatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.4 Flowcytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.5 Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.6 Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3 Results 40
3.1 CharacterisationofT-cellphenotypesinpatientswithmildandmoderateAD . . 40
3.1.1 Mild AD is associated with dramatic shifts in circulating CD4 but not
CD8T-cellsubsetsrelativetoage-matchedcontrols . . . . . . . . . . . . 40
3.1.2 Frequency of naïve, late-differentiated and regulatory T-cells in the pe-
ripheralbloodofpatientswithmildandmoderateAD . . . . . . . . . . 41
3.2 ImmunesignaturesofCanadianpatientswithmild,moderateorsevereAD . . . 47
3.2.1 T-cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.2.2 Tregs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.2.3 Chemokine receptor expression on the leukocytes of AD patients and
healthycontrols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.2.4 InducedcytokineproductionprofilesofCD4+T-cellsinADpatientsand
healthycontrols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.3 ImmuneprofileofItalianpatientswithAD,VaDormixeddementia . . . . . . . 58
3.3.1 T-cellsandtheirphenotype . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.3.2 RegulatoryT-cellsandchemokinereceptors . . . . . . . . . . . . . . . . 63
3.3.3 Leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.3.4 Amyloidbetauptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.3.5 Cytokineprofile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
3.3.6 CMVserostatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.4 ComparisonofrpAD,MCI,ADandhealthyimmunestatus . . . . . . . . . . . . 69
3.4.1 T-cellphenotypesinADpatientsandhealthycontrols . . . . . . . . . . 69
3.4.2 Tregs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3.4.3 Otherleukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3.4.4 B-cellphenotypefrequenciesinADpatientsandhealthycontrols . . . . 76
3.4.5 Chemokinereceptorexpression . . . . . . . . . . . . . . . . . . . . . . 80
4 Discussion 84
4.1 T-cell phenotypes: a shift from early- to late-differentiated CD4+ T-cells in AD
patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.2 DistributionofregulatoryT-cellsubsetsinADpatients . . . . . . . . . . . . . . 89
4.3 LeukocytesinhealthycontrolsandADpatients . . . . . . . . . . . . . . . . . . 90
4.3.1 ControversialresultsconcerningthefrequencyofT-cellsinAD . . . . . 90
III
4.3.2 Reduced frequencies of naïve B-cells in AD patients compared with
healthycontrols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
4.3.3 NodifferenceinthefrequencyofNK-andNKT-likecellsinADpatients 93
4.3.4 No significant differences in the frequency of monocytes between de-
mentiapatientsandhealthycontrols . . . . . . . . . . . . . . . . . . . . 93
4.4 Degree of T-cell cytokine production does not differ between AD patients and
healthycontrols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
4.5 Higher chemokine receptor expression in AD patients compared with healthy
controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
5 Summary 101
5.1 SummaryinGerman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Glossary 124
IV
Chapter 1
Introduction
1.1 Types of dementia and their diagnosis
Dementiaisdefinedasalossofmemoryandothercognitivefunctionsrelevanttodailylifewhich
persists for more than six months. The cause of the disease is the destruction or death of brain
cells. Inorderfordementiatobediagnosed,atleasttwoofthefollowingfourcognitivefunctions
shouldbeimpaired:
"(1) memory; (2) ability to speak or understand language; (3) capacity to plan,
make sound judgments, and carry out complex tasks; and (4) ability to process and
interpretvisualinformation"
[70]. ClinicallydementiacanbeclassifiedintodifferentcategoriesshowninFigure1.1.
Dementia with
Parkinson,
Lewy bodies
Frontotemporal dementia,

HIV encephalopathy

Vascular dementia

MIXED dementia
Alzheimer disease
Figure1.1: Formsofdementia;afterBosser,1992(modified)[17]
1
Table1.1: Riskfactorsfordementia
categoryofdementia factor reference
cognitivedecline sex [1]
cognitivedecline age [1]
cognitivedecline education [1]
dementia income [1]
PD3 nocigarettesmoking [97]
neuronaldamage noconsumptionofnicotineandcaffeine [97]
cognitivedecline chronichealthconditions [1]
cognitivedecline inflammatorymarkers(CRP2,cytokines) [1]
AD,PD3 nononsteroidalanti-inflammatorydrugs [97]
AD,PD3 infections(influenza? Borelia? others?) [97]
cognitivedecline1 serumantibodiestoCMV,HSV-1,HSV-2 [146]
cognitivedecline maybehighanti-CMVIgGantibodylevels [1]
cognitivedecline HSV-1+positiveforApoE-E4allele [1]
AD ApoE-E4genemutation/ApoEstatus [70]
AD PS1/PS2genemutation [58]
AD familyhistoryofAD [70]
AD DownSyndrome [70]
PD3 excessweight/bmi4inmidlife [97]
AD obesity [70]
PD3 highcaloricintake [97]
AD hypercholesterolemia [70]
AD highhomocysteine [70]
AD hypertension [70]
AD type2diabetes [70]
AD metabolicsyndrome [70]
AD heartdisease [70]
AD cerebrovasculardisease [70]
AD folateorotherB-vitamindeficiencies [70]
1 inelderlyFinishwithvasculardisease
2 C-reactiveprotein
3 Parkinson’sdisease
4 bodymassindex
2
1.1.1 Mild cognitive impairment (MCI) and the difficulties of its diagnosis
Mildcognitiveimpairment(MCI)isabraindisorderassociatedwithadeclineincognitivefunc-
tion that does not interfere with daily life [90]. It is considered a transitional state that in many
cases leads to dementia [9]. There are many subtypes of MCI with amnestic MCI (aMCI) be-
ing the most prominent. aMCI is diagnosed when cognitive decline is accompanied by memory
impairment [112]. Both types can, but must not necessarily, progress to the different types of
dementia, including Alzheimer’s Disease (AD) [53, 174, 43]. Dubois et al. studied a cohort
of people with amnestic mild cognitive impairment. Of those which - according to the criteria
of that time - were clinically identified to have progressed to dementia only 70% met the neu-
ropathological criteria for AD. Therefore a question is posed as to when MCI can be considered
tohaveprogressedtoADandwheretodrawthedefininglineinwhatappearstobeacontinuous
process [32]. In order to identify predictive markers for this progression, Tabert and colleagues
analysed 148 patients and 63 age-matched controls in a follow-up study. The main conclusion
was that "deficits in verbal memory and psychomotor speed/executive function abilities strongly
predicted conversion to AD" [148]. Other risk factors for dementia are listed in Table 1.1. For
diagnosticvisualisationoftheconversionfromMCItoAD,MRI(MagneticResonanceImaging)
patterns of hippocampus atrophy were proposed [165] or in a more recent study a "Short-form
Everyday Technology Use Questionnaire" that can be used as a screening tool for evaluating
the use of everyday technology (e.g. using a telephone or household equipment, managing fi-
nances and transportation) [72]. Using MRI it is possible to analyse loss of grey matter, while
questionnaires can assess memory function, but neither technique is able to explain the origin
of disease. Another idea discussed in this context is the use of biological markers, such as spe-
cificimmunesignatureswhichmaycontributetounderstandingthediseaseprocessitself. Inthis
context, the present work was performed with the hypothesis that Cytomegalovirus (CMV) and
dementia(primarilyAD)influencetheimmunesystem. CMVinfectionhasbeenimplicatedasa
potential risk factor for AD (Table 1.1). It has a major impact on immune signatures which may
be relevant to MCI and in determining which patients with MCI may progress to AD. Earlier,
more accurate diagnoses would allow earlier therapeutic intervention which might increase the
cognitivelyhealthytimeperiodofaffectedindividuals.
1.1.2 Alzheimer’s Disease (AD)
FirstreportsonAD
Alzheimer’s Disease is the most common form of dementia, it occurs predominately in elderly
peopleabove65yearsofage. Accordingtothe2007FactsandFiguresreportoftheAlzheimer’s
AssociationoftheUnitedStates,96%ofallADpatientsareover65yearsofage. Theworldwide
prevalenceofADwasestimatedtobe18millionin2008[70],withapredictedincreasingpreva-
lenceduetorisinglifeexpectanciesandareductionindeathsfrominfectiousdiseasesassociated
withadvancesinmodernmedicine. Becauseofthis,thestudyofdementiaisbecomingmorerel-
evanttosocietalhealthandtheburdenofdisease. ADwasfirstdescribedbyAloysiusAlzheimer
in the beginning of the 20th century. Alzheimer observed memory decline, disorientation and
3
confusion in a 51 year-old patient in 1901 [70]. After autopsy in 1906, Alzheimer described
the main characteristics of AD: an atrophied brain, dead and dying brain cells, plaques consist-
ing of amyloid beta and neurofibrillary tangles (NFT) [5]. This led eventually to the "amyloid
hypothesis".
Amyloidbetaandtau
DespitecontroversialissuessurroundingthecauseandclassificationofAD,formationofamyloid
plaques remains the main diagnostic and causative factor. It has been suggested by the group of
LaFerlathatamyloidbeta(Aβ)maychangefrombeingabeneficialmoleculewithantimicrobial
activity that is involved in protection against oxidative stress, cholesterol transport and is im-
plicated in certain signalling pathways, to one of the major candidate molecules suggested to
contribute to AD pathology (Fig. 1.2). The amyloid precursor protein (APP) is processed in
cholesterolrichmembraneregions,so-calledlipidrafts. SecretasescleaveAPPintoproteinsbe-
tween 36 and 43 amino acids long. The most important forms for AD pathology are suggested
to be Aβ-40 and Aβ-42. Aβ can accumulate and aggregate, which according to the amyloid
hypothesis initiates the onset of AD [117]. It is believed that Aβ exerts negative (for exam-
ple neurotoxic) effects and has an immunomodulatory role. Synaptic dysfunctions within the
brain and a systemic inflammation are some of the consequences of Aβ accumulation. With mi-
croscopy studies the La Ferla group showed co-localisation of ubiquitin and Aβ, implying that
proteasomalfunctionsareinvolvedintherolethatAβ mayplayinADpathology. Onetargetfor
proteasomal degradation is phosphorylated tau, which can accumulate and form neurofibrillary
tangles, as observed by Alzheimer within the brain of one of his patients. Tau is a microtubule-
binding protein, which is involved in axonal transport, also of APP, and can so influence Aβ
distributionand/orproduction. AnotherhypothesisisthatAβ activatestaukinaseswhichleadto
hyperphosphorylation of tau. The consequence is dissociation from microtubuli which then de-
polymerise, which results in axonal transport deficits and accumulation of hyperphosphorylated
tauinADpatients. Additionally,consistentwithastateofsystemicinflammation,microgliaand
astrocytes are activated and are located near Aβ plaques, - as for example shown by La Ferla.
These cells possess phagocytic activity and degrade Aβ plaques. In addition they produce high
levels of pro-inflammatory cytokines, which can activate certain kinases, leading to tau hyper-
phosphorylation[13].
AmyloidbetaasthecauseofAD-acontroversialhypothesis
Among several theories suggested to explain the cause of AD, the amyloid beta hypothesis is
the most prominent. In 1991 Hardy and Allsop proposed a sequence of events characteristic
of AD. Due to an imbalance between production and clearance of Aβ, the primary component
of the plaques seen by Alzheimer, there is an accumulation of Aβ. This leads to formation of
neurofibrillary tangles and subsequent to neuronal death [57]. This hypothesis is supported by
the fact that the gene for the Aβ precursor protein (APP) is located on chromosome 21 and that
individuals suffering from Down Syndrome that possess an extra copy of chromosome 21 fre-
quently develop AD [89]. In addition, mutations in the APP gene could cause Aβ accumulation
4