MethodologicalChallengesinCharacterizingGnotobiotic AnimalModels:

Reproducibility:Withinandacrossexperiments

Theroleofimmunologicvariation

Jeremiah Faith
Icahn School of Medicine at Mount Sinai
Immunology Institute
Institute for Genomics and Multiscale Biology
ILAR: Animal Models for Microbiome Research
Dec 20, 2016

Differencesinmicrobiome driveimmunologicvariation
Jacksonvs Taconicmicrobiota isourmostwidelyusedanddistributedcomparison
SFB

Jax

Jax +SFB

IL17

GF

CD4

Ivanov,et.al.,Cell2009

Sivan,et.al.,Science2015

RelativeAbundancevs.AbsoluteAbundance

EduardoContijoch

Absolutegut
microbiotaabundance

Relativegut
microbiotaabundance

+
Biomass
(ug DNA/mgfecalsample)

Absolutemicrobiotaresponsetoantibiotics
Vanco

Akkermansia sp.
(vancomycin resistant)

Absolutegutmicrobiotaabundance

Biomass(ug DNA/mgfecalsample)

Absolutemicrobiotaresponsetoantibiotics
nondepletingantibiotic

Vancomycin

Absolutemicrobiota variesbyJacksonlabsanimalbatch

Akkermansia sp.
(vancomycin resistant)

Vancomycin absolutebloom
Absolutegutmicrobiotaabundance

Absolutegut
microbiotaabundance

Vancomycin absolutedepletion

TwosetsofC56Bl6micefromJacksonlabstworesponsestoantibiotics

Concernsforanimalwelfare
Lackofdefinedcommunitiestostudyimmunologic
variation
Currentstandard~=Jacksonmicrobiota vs Taconic
theseareNOTstandardizedordefined!

Microbiota immunevariationacrossanimalfacilities,
betweenrooms,acrosstimeconfoundsresults
Ifwecannotrepeateachothersfindings(orourown),are
wedoingscience?

Highthroughputgnotobiotics
Traditionalgnotobiotics
onemicrobiota =oneisolator

Highthroughputgnotobiotics
onemicrobiota =onecage

Newdevelopment:
HEPAfilteredpositivepressurecages
Mostimportantfactorisstilloperatortraining

Identifyinghumangutmicrobialinfluencesin
gnotobiotic mice

GrahamBritton

Repeatabilitywithstandardizedfecalmicrobiotas

Microbialloadandcommunitychangesinmicrobesare
similaringermfreeandcolonizedanimals

Repeatabilitywithmanufacturedculturecommunities

Anaerobicisolation

Proteinextraction

Identification
(MALDITOF)

Currentthroughput
1library/week(20species/40strains)

Donor

Goodman,et.al.,PNAS 2011
Faith,et.al.,Science2013

Eachwellcontainsaunique
bacterialstrain

Humangutmicrobestransfercolitistosusceptiblemice
CD45RBHI T cell transfer colitis model
Reconstitute with
CD45RBHI CD4+ T cells

Monitor loss in body weight to

assess colitis severity

Personalized gnotobiotic Rag-/- mice

microbiota of known colitogenicity included

in every experiment

Ulcerativecolitis

Variation from colonization with other

human microbiotas

Interpersonal human gut microbiota variation drives:

diverse Treg in unchallenged mouse models

Humanmicrobiotas

Interpersonal human gut microbiota variation drives:

diverse Th17 in unchallenged mouse models

Humanmicrobiotas

Influenceofinterpersonalhumangut
microbiota variationonhosthealth

Identifyingeffectorstrains

Screeninghumanizedmice
Diversedonors

Gnotobiotic mice

%FoxP3+among
CD4+Tcells

Humanizedwiththe
microbiota ofauniquedonor

Whichbacteriaare
causingthis?!?

Faith,Ahernet.al.,Sci Transl Med2014

Largescalebacterialisolation
Eachwellcontainsaunique
bacterialstrain
(17totalmicrobes)

Donor

Fractionatecommunity

Gnotobiotic mice

Communitysize

Adiposity

Serine

%Tregs

Phenotypescreen

Phenotyperesponse

Treg inductionbydefinedcommunities

AlteredSchaedler flora
(8mouse gutmicrobes)
Geuking,et.al.,Immunity2011

Whichbacteriaare
causingthis?!?

%FoxP3+among
CD4+Tcells

%FoxP3+among
CD4+Tcells

Identifyingeffectorstrains

Faith,Ahern,et.al,Sci Transl Med2014

Identifyinganimmunomodulatory
effectorstrain
ElevatedTh17notcommon
Effectorstrain(s)notcommon

Combinatorial gnotobiotics as a tool to identify effector strains within a complex

defined microbiota

Community

IL17A
(%ofliveCD4+)

Identifyinganimmunomodulatory
effectorstrain

Enterobacteriaceae 1

Two validation approaches with defined microbiotas

Whole cultured community,

removing Enterobactericeae 1
or Enterobactericeae 2

em

be

r+

be

A6

em

R.gnavus,C.boltae,C.aerofaciens,B.vulgatus)

7m

(*B.thetaiotaomicron,B.ovatus,B.caccae,

p=0.012

7m

7-member model community*

With addition of Enterobactericeae 1

% IL-17A+
of live CD4+

Challenges:creatingscalablesystemsforthefuture

SPF
SOPs:designedtokeepknownpathogensout

DefinedMicrobiotas
SOPs:designedtomaintainconsortia
Derivedfrom~10human/mousemicrobiotas

Effectorstrains
Individualorgroupsofmicrobeswithknownfunction
Knownabilitytoengraftondefinedmicrobiotas
Canbeusedtomodulatedefinedmicrobiotas

Buildsystems(governmentsuppliers,commercialvendors,
internalSOPs)tostandardizemicrobiotas acrosstheworld

Thanksto
Ourlaboratory
JoshBorgerding [jointwithMerad]
GrahamBritton
EduardoContijoch
RubenDorador
GrantEscano
Cinkia Fermin
Zhihua Li
SeanLlewellyn
Ilaria Mogno
EddieVazquez
OliviaVennaro
ChaoYang

Collaborators(IBD)
MarlaDubinsky[MountSinai]
JonathanJacobs[UCLA]
JonBraun[UCLA]
JeanFredericColombel [MountSinai]
JoseClemente[MountSinai]
AriGrinspan [MountSinai]
JudyCho[MountSinai]
IngaPeter[MountSinai]
BruceSands[MountSinai]
SergioLira[MountSinai]
JoanaTorres[MountSinai]
JeffreyGordon[WashU]
PhilipAhern[WashU]
(Cancer)
MiriamMerad [MountSinai]
JamieFerrara[MountSinai]
RandallHolcombe[MountSinai]
CelinaAng [MountSinai]

Immunology Institute
Institute for Genomics and Multiscale Biology

Icahn School of Medicine at

Mount Sinai

(Animals)
CrystalJohnson[MountSinai;CCMS]
Sheela Hira [ZooKnoxville]

(HIMC)
Seunghee Kimschulze [MountSinai]
Xiaochen Qin(Ivy)[MountSinai]
Hui Xie (Emily)[MountSinai]

(FMTcoordinators)
Tramy,Bridget,Phil

Funding
NIH/NIGMSGM108505
NIH/NCCIHAT008661
NIH/NCICA039542
SUCCESS
CCFAMicrobiome Innovation
NewYorkCrohns Foundation
Janssen Human Microbiome Institute
JanssenRTP
JanssenMIND

Immunology Institute
Institute for Genomics and Multiscale Biology

Icahn School of Medicine at

Mount Sinai