regulating immune responses to viral infection
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Regulatingimmuneresponsestoviralinfection
DonnaMacDuff,PhD
AssistantProfessorDepartmentofMicrobiologyandImmunology
dmacduff@uic.edu
MicrobeGrowth
UncontrolledReplication ImmunePathology
PathogenClearance
Howistheimmunesystemregulated?
MolecularmechanismsPhysiologicalrelevance
AnimalmodelsKnock-outmice
CellculturesystemsBiochemicalapproaches
Bacteria Viruses
CommonExperimentalApproachestoInterrogatetheImmuneSystem
Boisson etal.2012
WhyareHOIL1andLUBACsoimportantfortheimmunesystemtofunctionproperly?
HOIPattaches“linear”ubiquitinchainstootherproteinsHOIL1isalsoaubiquitinligase,butitsfunctionisunknown
Potentialfunctionsforubiquitinchainsattachedtoproteins:Facilitateproteininteractions/actasascaffoldMarkproteinsfordegradationbytheproteasomeActivateorinhibitproteinfunction
LUBAC=LinearUBiquitin chainAssemblyComplex
Linearubiquitinchainassemblycomplex(LUBAC)
Toll-likereceptorsInflammatorycytokines
Necroptosis RIG-I-likereceptorsInterferons
NOD-likereceptorsInflammasomePyroptosis
RodgersMAetal.,JEM2014InnKSetal.,MolCell.2011KhanMetal.,PLoSPathog.2016ZhangMetal.,CellResearch2008BelgnaouiSMetal.,CHM2012
ChattopadhyaySetal.,Immunity2016LiuSetal.,eLIFE2013
TokunagaFetal.,NatCellBiol.2009HaasTLetal.,MolCell.2009DamgaardRBetal.,MolCell2012HostagerBS,etal.,PloSOne2010ZakDEEtal.,PNAS2011others
ImmunefunctionsofLUBACandlinearubiquitination
LUBACregulatestheimmuneresponseaftersensingofpathogens
HOIL-1-/-
Highlysusceptibleto: Moreresistantto:
MacDuffDAetal.,eLIFE 2015
WhatarethephysiologicalfunctionsofLUBAC?HOIL-1-deficientmice– modelforhumandisease
Hyper-inflammatory?Intestinalabnormalities?
Immunodeficient?Cardiomyopathy?Murinenorovirus
Listeriamonocytogenes
Toxoplasmagondii
Mycobacteriumtuberculosis
Murinegamma-herpesvirus68
Citrobacter rodentium
MacDuffDAetal.,JVI2018
Currentstudiesinthelab
Commensalmicrobes
Type2inflammationintheintestine
Citrobacter rodentium(modelforE.coli)
SurvivalIntestinalbarrierintegrity
Murinenorovirus
TypeIandIIIInterferons
MDA5(notRIG-I)MacDuffetal.,JVI2018
HowdoesHOIL1balanceinflammationandinnateimmunitytocommensalandpathogenicmicrobesintheintestine?
On-goingstudiesinthelab
§ Closelyrelatedtohumannorovirusesthatcauseacutegastroenteritis
§ Endemicinmany(most)mousefacilitiesworld-wide
§ (+)ssRNA,non-envelopedvirus(Caliciviridae)
§ Infectslaboratorymiceinvivobygastro-intestinalinfection
§ Replicatesinmacrophagesanddendriticcellsinvitro
§ Interferonsareimportantforcontrollingacuteandpersistentinfection
Murinenorovirus(MNoV)
MurinenorovirusreplicatestohigherlevelsinHOIL1-deficientmice
10 20 302
3
4
5
6
7
Days after MNoV infection
Log 10
MN
oV c
opie
s/fe
cal p
elle
t
Hoil-/-Hoil+/+
WT Hoil1-/- WT Hoil1-/- WT Hoil1-/-
2
4
6
8
Log 10
MN
oV c
opie
s/ug
RN
A(n
orm
aliz
ed to
Rps29
)
Ileum Colon MLN
**** *
Viralsheddinginstool Viralburdensintissues
14dayspost-infection
B6
Hoil1-/-
0
1
2
3
4
IFNb transcript induction9 hpi MNV CR6 MOI = 3
log1
0 IF
Nb
fold
indu
ctio
n re
lativ
e to
WT
unin
fect
ed
HOIL1-deficientbonemarrow-deriveddendriticcellsmake100Xlessinterferon-betamRNAafterinfectionwith murinenoroviruscomparedtowild-typecells
~100x
IFN-bmRNA
HOIL1isimportantforinducinginterferonsduringmurinenorovirusinfection
WTHOIL1-/-
Howarevirusessensedbycellstoinduceinterferons?
Interferonsmadebyinfectedcellsactivateneighboringcellstoarmthemselvesagainstinfection.TheviralRNAsensor,MDA5,isimportantfortheinterferonresponsetoMNoV:“MDA-5recognitionofamurinenorovirus.”McCartneySAetal.,PLoS Pathog.2008
HallerOetal.,Virology2006
MNoVSeV
SeV (0
.1X)
VSV0
1
2
3
4
5
log1
0 IF
Nb
fold
indu
ctio
n re
lativ
e to
WT
untr
eate
d ControlHoil1-/-Mda5-/-
MNoVTMEV
poly(I:C
)
poly(I:C
) txn
0
1
2
3
4
5
log1
0 IF
Nb
fold
indu
ctio
n re
lativ
e to
WT
moc
k
ControlHoil1-/-Mda5-/-
IFN-b******
********
**
******
Bonemarrow-deriveddendriticcells
HOIL1isrequiredduringinfectionwithvirusessensedbyMDA5butnotwithvirusessensedbyRIG-I
10-100-folddefectsinIFNb mRNAinductionafterMNoV,TMEVinfectionorpoly(I:C)RNAtransfection– detectedbyMDA5.NodefectsinHOIL1-deficientorMDA5-deficientcellsafterinfectionwithSeV orVSV-detectedbyRIG-I.
MNoV =murinenorovirusSeV =SendaivirusVSV=vesicularstomatitisvirusTMEV=Theiler’smurineencephalomyelitisviruspoly(I:C)=syntheticdouble-strandedRNA
RNA
Interferonsa,b,l
ligand
receptor
adaptor
kinaseregulator
TranscriptionFactor
Transcript(mRNA)
SeV,VSVRNA
MAVS
IRF3/7
MDA5
TBK1/IKKeTANK/NEMO
HOIL1/LUBAC?
MNoV,TMEV
RIG-I?
HOIL1isrequiredforinductionofinterferonsdownstreamofMDA5,butnotRIG-I.ButMDA5andRIG-Iuseacommonsignalingpathway
HowdoesHOIL1regulatethesesignalingpathwaystocontrolviralinfection?
HOIL1/LUBAC?
• IdentifyproteindomainsofHOIL1(andHOIP,Sharpin)thatareimportantforinterferonproduction(generateandtestmutantgenes)
• IdentifyHOIL1-interactingproteins(testcandidateinteractors,developasystemtoidentifynewinteractors)
• Identifyproteinsthatareubiquitinated byHOIL1(testcandidateproteins,developasystemtoidentifyubiquitinated proteins)
• TestwhetherHOIL1activityalterspost-translationalmodifications(phosphorylation,ubiquitination,etc)onMDA5
• TestwhetherHOIL1expressionisimportantinmacrophages,dendriticcellsorintestinalepithelialcellsduringviralinfectioninvivo(infectmutantmiceandmeasureviralburdens)
PotentialRotationProjects
Questions? dmacduff@uic.edu
Labwebsite: https://sites.google.com/uic.edu/macduff/home
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