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Evolving an established

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

Elements fostering sustained success


Mission and cost structure that matches users
needs
Sustained, broad- based funding
Stable leadership (scientific and technical)
Staff continuity
Committed core user base
Transparent and equitable prioritization
Adequate facilities for expansion and upgrades
Continuously upgrade services, resources and
quality of science

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.

The aims of the NGRRC are to:


1. Provide axenic (germ free, GF) or gnotobiotic mice for NIH, Center for
Gastrointestinal Biology and Disease (CGIBD) or Crohns and Colitis Foundation
(CCF) funded investigators investigating diverse host/ microbial interactions.
2.DeriveadditionalGFgeneticallyengineeredmousestrains.
3.Supportpilotstudiesforinvestigatorswithnovelhypothesestogenerate
preliminarydataforNIHgrantapplications(prioritizingyoung/newinvestigators).
4.Trainpersonneltodevelopgnotobioticfacilitiesatotherinstitutions
5.Developinnovativetechniquestoimprovegnotobiotic resources(Research
Component)

History of Rodent Gnotobiotic facilities at


NCSU- Vet School and UNC
Founded at NC State Univ., College of
Veterinary Medicine 1985 by Phil Carter as core
facility for the CGIBD (P40 NIDDK)
Parallel facility founded at UNC-Chapel Hill
2001 to accommodate expanded user base,
provide backup of critically important GF strains,
partnership UNC MMRC
Creation of National Gnotobiotic Rodent
Resource Center- 2004 external funding by
Comparative Medicine Section, NCRR (NIH)

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)

Advocating for institutional support of a


gnotobiotic unit
Increase competitiveness for NIH and
foundation grant applications (innovative
mechanistic and functional studies)
Tool for recruiting talented faculty who will
bring and generate more grant support
Increase national visibility and reputation
Cutting edge research technology

Growth of National Gnotobiotic


Rodent Resource Center
1985
Isolators
6
Users
3
Mice/ rats
?
Strains:
Mice
0
Rats
1
Zebrafish

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

Germ-free mouse breeding stocks at UNC


IL-10 deficient (129 S6 SvEv)

129 S6 SvEv wild type

IL-10-/- x Rag 2-/- C57 BL/6J

Rag 2-/- 129 S6 SvEv

C57Bl/6J

IL-10-/- x NFBEGFP transgenic*

BALB/c

RP 105-/- (C57 BL/6)

Swiss Webster

Sccn1b-TG

IL-10EGFP transgenic

IRGM-/- (C57 BL/6)

TLR 5-/- C57 BL/6

NOD SCID IL-2R-/- (NSG)

ATG 16L1 hypomorph

NF IL-3-/- (C57 BL/6)*

IL-10-/- x Rag 2-/- 129 S6 SvEv

IFN-/- (C57 BL/6)*

NOD 2-/- (C57 BL/6)

TL1A-/-

AGR2-/+ (mixed)

NFBEGFP transgenic*

ATG16L1T300 KI (human TG)

APCMIN/IL-10-/-

IL-10-/- (C57 BL/6)

PI3K (C57 BL/6)*

P48Cre/Kras

ACT-/+

DRTSV28+/-

PlgR-/- C57 BL/6J

NLRP12-/-

Sox 9 reporter

NLRC3-/-

Elements fostering sustained success

Stable leadership (scientific and technical)


Staff continuity and feeling of ownership
Committed core user base
Transparent and equitable prioritization
Adequate facilities for expansion and upgrades
Continuously upgrade services and resources

Secret to our success


Maureen Bower, LATG
Technical Director
National Gnotobiotic Rodent Resource Center
University of North Carolina Chapel Hill

Recruited to UNC after retiring after 20 yrs. at


Taconic Farms as Manager, Gnotobiotic Facility.
Vast experience in gnotobiotics, training UNC
staff.
Established hands- on 1 week training program
for external facility directors and staff wishing to
start their own gnotobiotic facilities

Encouraging staff stability and


professional growth
Realize that this is exacting and repetitive work
that requires extensive training- merits higher
pay scale and classification than standard mouse
care provider. More than cleaning cages!
Provide upward mobility with increased pay
scales/ promotions and learning more extensive
techniques (gavage, IP injection, genotyping,
breeding strategies, etc.)
Have investigators return to describe results,
why technicians work is important, what was
accomplished

Elements fostering sustained success


Committed core user base
Transparent and equitable prioritization
Established prioritization committee and a webbased scoring system with points for prioritization
(NIH or CCFA funding, CGIBD member, young/
new investigator, time on waiting list, need urgent
data for pending manuscript or grant). Scores and
mouse availability determine access.
Adequate facilities for expansion and upgrades
Continuously upgrade services and resources

Customized surgical and GF derivation isolator

Evolution of scientific uses and technologies


GF vs. SPF (SPF, conventionally raised), GF
SPF
Timing of colonization- age dependency, kinetics of host
responses
Mono dual or defined group association fecal
transplant (mouse or human, different phenotypes)
Bacterial strain specific responses
Explore functions- bacterial or host genes- KO, TG mutant
Gene expression- host, bacterial under different conditions
Humanized- human fecal transplant, human immune
response (NSG +/- human MHC), human diet
Metabolomic studies
Bacterial adaptation to different environments

Insights into microbial function from gnotobiotic rodents


National Gnotobiotic Rodent Resource Center (NIH, CCFA)

The stage:

A gnotobiotic
isolator

The actors:
Germ-free
(monoassociated)

Selectively SPF/ CONV-R

colonized

SPF raised

CONV-D

conventionalized

Adapted from Jeff Gordon

Essential Role of Commensal Enteric Bacteria in the


Pathogenesis of Experimental Chronic Intestinal
Inflammation
No bacteria

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

Differential ability of various bacterial species


to induce or prevent experimental colitis

All bacterial species are not equal!

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

Host-specific responses to selected enteric


commensal bacterial species (Sandy Kim 2005)
Host
HLA B27 TG rat
.

Monoassociated bacteria
Bacteroides vulgatus
E. coli
Enterococcus faecalis

IL-10 deficient mouse

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

Different E. coli strains induce variable


degrees of colitis in monoassociated IL-10-/mice
4

*p < 0.02
129 SvEv
IL-10-/-

*p < 0.03

Histology score

0
NC101

Sandy Kim, et al., 2008

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

Determining E. coli Transcriptional


Responses to Intestinal Inflammation
Jonathan Hansens lab

GF IL-10-/-

E. coli NC101

GF WT

Patwa LG et al, Gastroenterology, 2011

Upregulation of E. coli Stress


Response Genes in Colitis
Description

7.3

ibpB

Heat shock protein

4.7

ibpA

Heat shock protein

5.3

glpC

Anaerobic glycerol-3-phosphate dehydrogenase

4.4

oxyS

Global regulatory RNA element

5.1

gadA

Glutamate decarboxylase isoenzyme

4.4

gadB

Glutamate decarboxylase isoenzyme

2.3

yhiX

Transcription factor for gadA/B

3.2

hdeB

Hypothetical protein (acid resistance)

2.8

hdeA

Hypothetical protein (acid resistance)

p 0.05
All genes vs WT

Acid Stress

Gene Name

Oxidative Stress

Fold Increase

Selection of Bacteria for a Simplified Human Microbiota


(SIHUMI) Relevant to IBD
Selection criteria:
identified to play a positive/negative role in experimental colitis or
differentially regulated in IBD
human origin
genome sequence available
capable of colonizing the murine intestinal tract to form a stable community
Phylum

Species

Strain

Proteobacteria

Escherichia coli

LF82

Firmicutes

Faecalibacterium prausnitzii

A2-165 (DSM 17677)

Firmicutes

Enterococcus faecalis

OG1RF

Actinobacteria

Bifidobacterium longum
subsp. longum

DSM 20219 (ATCC 15707)

Firmicutes

Lactobacillus plantarum

WCFS1 (ATCC BAA-793)

Bacteroidetes

Bacteroides vulgatus

DSM 1447 (ATCC 8482)

Firmicutes

Ruminococcus gnavus

ATCC 29149

SIHUMI induce colitis in selectively colonized gnotobiotic


IL-10-/- mice but not wild type 129 SvEv mice
12
ChangSoo Eun et al, Infection & Immunity, 2014
P=0.002

Histology score (0-12)

P=0.026

129 WT

B6 WT

129 IL10

B6 IL10

12 weeks

129 IL10(6w)B6 IL10(6w)

6 weeks

Selective Induction of IFN- in MLN Supernatants by Individual


Bacterial Lysates in 129 IL-10-/- and B6 IL-10-/- mice
after SIHUMI Colonization
Control

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)

129 (12 wks)

B6 (12 wks)

EC, E.coli; EF, Enterococcus faecalis; RG, Ruminococcus gnavus; BV,


Bacteroides vulgatus; FP, Faecalibacterium prausnitzii; BL,
Bifidobacterium longum; LP, Lactobacillus plantarum

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

Fold change (vs.GF)

II. Regulatory cytokine expression


RT-PCR (distal colon)
Il17a
8

Rort
100

* Ifng

* *

Tbet

80

60

40

20
0

InductionofIL10by
commensalintestinalbacteria

IL10GF IL-10+/EGFP reporter mice

YoshiMishima(DDW2012)

Colonized with SPF


feces
0 (GF)

SPF-raised

Commensal intestinal bacteria up-regulate mucosal IL-10


Colonic tissue culture (IL-10,
ELISA)

IL-10 secreting cells (FACS)

IL10

# of GFP+ cell / 106


colon LP cells

IL-10 (pg/ml/50mg tissue)

***

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

IL-10 mRNA in distal colon


(PCR)

28

***p<0.001, **p<0.01, *p<0.05, vs. GF

Kinetics of bacteria-induced RNA profiles in the distal colon


Il10+/+ mice
Il10-/- mice
***
***
** *
*
***
*
*
*

******
**
**

*** ** *
*

**

***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

# of GFP+ cell / 106 colon LP cells

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

Cornell Vet School


Kenny Simpson
Tokyo Univ.
Kenya Honda
NIH grants P40 OD010995, P01 DK094779,
R01 DK053347, P30 DK034987, CCFA, K08
DK063111 (Kim), 5KL2RR025746 (Gulati)
Young Investigator- Probiotics (Gulati)