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gnotobiotic facility
R. Balfour Sartor, M. D.
Professor of Medicine, Microbiology &
Immunology
Director, Multidisciplinary IBD Center and
National Gnotobiotic Rodent Resource
Center
University of North Carolina
NationalGnotobioticRodentResourceCenter
The National Gnotobiotic Rodent Resource Center (NGRRC) funded by NIH ODs (NCRR)
Comparative Medicine Section since 2004 provides a resource for local, regional, national
and international multidisciplinary investigators to explore the hypothesis that resident
bacteria fundamentally influence adaptive physiologic processes in normal hosts, but
induce pathogenic inflammatory, metabolic and neoplastic responses in susceptible hosts
with genetic risks or environmental (diet, infectious, etc.) triggers.
Funding Sources
CGIBD Core Facility (P40 NIDDK) since 1985
2001 expansion- NC Biotechnology Center, UNCCH Deans Office, NIDDK P40 supplement, CCFA,
used equipment from Univ. Wisconsin (Ed Balish)
2004 NIH P40 resource grant
2009 funding by CCFA as resource to IBD
investigators
2013 P01 NIDDK (core facility)
2016 R01 (Ian Carroll), startup (Janelle Arthur)
Program income- user fees ($30/ mouse, $300/
month isolator rental, $3,500 GF derivation)
2004
24
14
850
2008
40
39
1224
2013
78
35
2098
2016
100
36
2350
5
3
8
3
yes
29
0
yes
32
0
0
C57Bl/6J
BALB/c
Swiss Webster
Sccn1b-TG
IL-10EGFP transgenic
TL1A-/-
AGR2-/+ (mixed)
NFBEGFP transgenic*
APCMIN/IL-10-/-
P48Cre/Kras
ACT-/+
DRTSV28+/-
NLRP12-/-
Sox 9 reporter
NLRC3-/-
The stage:
A gnotobiotic
isolator
The actors:
Germ-free
(monoassociated)
colonized
SPF raised
CONV-D
conventionalized
No immune
activation
Mice
Resident bacteria
IL-2KO ()
IL-10KO
TNFARE
TCRKO
Macrophage
CD326TG
and TH1/TH17
KO
MDR1
immune
SAMP1/Yit ()
activation
hi
CD45RB SCID
Rats
HLA-B27 TG
Indomethacin
No colitis
Guinea pigs
Carrageenan
Non-human primate
Cotton top tamarin
Colitis
HLA B27
transgenic
rat
Aggressive
colitis
Cecal
bacteria
Bacteroides
vulgatus
Germ
free, no
colitis
Cecal bacteria +
Lactobacillus GG
Rath et al., J Clin Invest 1996;98:945
Rath et al., Infect Immunity 1999; 67:2969
Dieleman et.al., Gut 2003; 52:370
Moderate
colitis
No colitis
E. coli
Protection
Monoassociated bacteria
Bacteroides vulgatus
E. coli
Enterococcus faecalis
B. vulgatus
E. coli
.
E. faecalis
BM CD3 TG mouse
B. vulgatus
E. coli
E. faecalis
Outcome
Colitis (cecal
predominant)
No inflammation
No inflammation
No inflammation
Colitis (cecal
predominant)
Colitis (distal),
duodenal obstruction
No inflammation
No inflammation
No inflammation
*p < 0.02
129 SvEv
IL-10-/-
*p < 0.03
Histology score
0
NC101
LF82
E. coli strain
K12
Colitogenic effectsofadherent/invasiveE.coli
(AIEC)mutantsinmonoassociatedIL10/ mice
(withKennySimpson,BelginDugan,CornellVetSchool)
Pathway
E.coli
Strain
Mutant
Host
Schedule
*Fucose:
CUMT8WT,fucA IL10/,WT?,mechanisms
Propanediol CUMT8WT,pduC IL10/,WTNodifference
Ironuptake NC101WTvs.fyuA IL10/,WTcolitis,cancer
IrontransportNC101WTvs.tonB IL10/.WT,willrepeat
*Ethanolamine CUMT8WTvs.eutH IL10/,WTcolitis
CUMT8WTvs.eutE IL10/,WTPlanned
Hypothesis:Health(E.coli):fucose >ethanolamine
CD(AIEC):Ethanolamine>fucose
Ironuptake/utilizationfostersgrowth,virulenceAIEC
DeletionofEutH decreasescolitisinEcoli
monoassociated IL10/ mice
Rectum
129 IL-10KO
Cecum
129 IL-10KO
12
p*=0.01
8
HistologyScore
p**=0.003
c
u
m
T
8
e
u
tH
c
u
m
T
8
c
u
m
T
8
e
u
tH
c
u
m
T
8
Histology Score
12
DeletionoffucApotentiatescolitisinE.coli monoassociatedmice
GF IL-10-/-
E. coli NC101
GF WT
7.3
ibpB
4.7
ibpA
5.3
glpC
4.4
oxyS
5.1
gadA
4.4
gadB
2.3
yhiX
3.2
hdeB
2.8
hdeA
p 0.05
All genes vs WT
Acid Stress
Gene Name
Oxidative Stress
Fold Increase
Species
Strain
Proteobacteria
Escherichia coli
LF82
Firmicutes
Faecalibacterium prausnitzii
Firmicutes
Enterococcus faecalis
OG1RF
Actinobacteria
Bifidobacterium longum
subsp. longum
Firmicutes
Lactobacillus plantarum
Bacteroidetes
Bacteroides vulgatus
Firmicutes
Ruminococcus gnavus
ATCC 29149
P=0.026
129 WT
B6 WT
129 IL10
B6 IL10
12 weeks
6 weeks
Mixture
EC
EF
RG
BV
FP
BL
LP
600,000
500,000
IFN
(pg/mL) 400,000
300,000
200,000
100,000
0
129 (6 wks)
B6 (6 wks)
B6 (12 wks)
N.S.
15
10
10
TGF-b1: possible?
300
200
100
C GF
lo
-H
u
H Hu
u
C -Cl
lo o
-E
ER
EE EE
R R
-C
lo
Foxp3
G
C F
lo
-H
u
H Hu
u
C -Cl
lo o
-E
ER
EE EE
R R
-C
lo
Tgfb1
* *
G
C F
lo
-H
u
H Hu
u
C -Cl
lo o
-E
ER
EE EE
R R
-C
lo
N.S.
C F
lo
-H
u
H Hu
u
C -Cl
lo o
-E
ER
EE EE
R R
-C
lo
lo F
-H
u
H Hu
u
C -Cl
lo o
-E
ER
EE E E
R R
-C
lo
15
C GF
lo
-H
u
H Hu
u
C -C l
lo o
-E
ER
EE EE
R R
-C
lo
Rort
100
* Ifng
* *
Tbet
80
60
40
20
0
InductionofIL10by
commensalintestinalbacteria
YoshiMishima(DDW2012)
SPF-raised
IL10
***
Total cell
40000
30000
**
***
**
20000
1.0E-5
10000
0.0E+0
F
SP
D7
D3
GF
SPF
3.0E-5
2.0E-5
GF
28
******
**
**
*** ** *
*
**
***p<0.001
** p<0.01
* p<0.05
**
**
**
**
**
20000
10000
2500
500
5000
D
7
SP
F
D
3
SP
F
7
D
SP
F
7
D
3
D
SP
F
7
D
3
D
N.S.
1000
500
0
SP
F
10000
1500
15000
500
***
1000
SP
F
20000
3
D
N.S.
1000
2000
100
1500
F4/80+CD11b+
3000
200
SP
F
7
D
3
D
F
G
N.S.
300
5000
CD8+CD3+
10000
N.S.
400
CD11c+CD11b+
15000
4000
CD25+CD4+
1000
500
***
Foxp3-CD4+ (Tr-1)
1500
7
SP
F
3
D
F
G
CD25-CD4+
20000
2000
**
mice : GF Il10+/EGFP
Total GFP+
IL-10-secreting
immune cells in the
colonic LP (FACS)
**
CD19+B220+
30000
***p<0.001
** p<0.01
* p<0.05
( vs. GF)
Exp.Design:Rag2/IL10/ recipientmiceinoculatedwithselectedhighandlowratio
ofIL10/IFN bacteria.1weekaftercolonization,recipientsweretransferredCD4/B
cellsfromGFVertXmice.Miceweresacrificedat8weekafteradoptivetransfer.
Histology
High(good)
Duodenum
Cecum
Cecum
4
histological score
RatioofIL10/IFN
**
3
2
1
ad
G
oo
d
ad
G
oo
d
Low(bad)
Duodenum
3
2
1
ad
ad
G
oo
d
oo
d
0
G
High+Low
(good+bad)
Histologic score
ChallengesforGnotobiotic Facilities
Meetingneeds:Requestsoutnumberresources,
especiallyGFderivationsnewstrainsanddifferent
permutationsofcomplexbacterialmixtures
Matchingcostswithusersabilitytopayforservices
Betterintegrationofresourcesbetweencenters
avoidredundancy,promotesharingofstrains,
centralizedcryopreservedembryos,centralized
registries
Developcentralizedbanksofvarioustissuesand
RNAseq datafromGFvs.SPFisogenicmice
Standardizationofdefinedbacterialconsortia
Investigatetranskingdom microbialinteractions
Developbettertechiques formetabolomicsstudies
withinisolators
Acknowledgements
UNC
Ajay Gulati
Sandy Kim
Janelle Arthur
John Hansen
Christian Jobin
Steffen Wohlgemuth
Mike Shanahan
Minoru Matsuura
Ian Carroll
Bo Liu
Lisa Holt
Jessica Allen
Yoshi Mishima
Chang Soo Eun
Aki Oka
Univ. Cinncinnati
Chris Karp
Technical Univ. Munich
Dirk Haller
Univ. of DArvonne
Arlette Darfeuille- Michaud
UNC-Microbiome Core
Andrea Ascarate-Perez