Post on 20-Jun-2020
Innate Immune Dysfunction Results in Altered Gut Microbiota, Colitis and Metabolic Syndrome
Matam Vijay-Kumar, Ph.D
Department of Pathology,Emory University, Atlanta GA
Supported by CCFA and NIH K01
TLRs sense microbial productsTLRs sense microbial products(Bar Code Readers)(Bar Code Readers)
TLR2
NF-κB
Nucleus
MyD88
NF-κB
MyD88
NF-κB
TLR4
Peptidoglycan LPS
Proinflammatory Gene ExpressionTNFα, IL-1β, IL-6, IL-8
MyD88
NF-κB
TLR5
Flagellin
Cytosol
Gut Microbiota:A Neglected Organ in Organ
Lumenal Contents(50% bacteria by wt.)
1. Large area (200 m2 ≈ tennis court)
2. 1014 bacteria,≈ 500-1000 distinct species
3. Bacterial genes: 1 millionHuman genes: 25,000
4. Functions comparable to Liver
5. Gut bacteria: Yours BFF: Best Frenemy Forever
Goal of Mucosal Immune System: 1. Expediently detect/clear pathogens2. Keep opportunists in-check. 3.This must be done while avoiding harm to beneficial microbes and host tissues.
Nutrients
I. TLR5 deficient mice develop spontaneous colitis
II. TLR5 deficiency results in metabolic syndrome
Innate ImmunityInnate ImmunityAcute InflammationAcute Inflammation
ILIL--88
S. typhimuriumS. typhimurium
FlagellinFlagellin
Y
NFNF--kBkB
TLR5TLR5
TJTJIBD?IBD?
Does this happen in vivo in IBD?Does this happen in vivo in IBD?
Commensal microbesCommensal microbes
Epithelial cell
Gewirtz AT 2001 J Immunol
T5KO mice do not exhibit innateT5KO mice do not exhibit innateimmune responses to flagellinimmune responses to flagellin
0 20 40 60 80 1000
2
4
6
8
Seru
m IL
-6 [n
g/m
l]
Flagellin [µg/mouse]
WT littermate
T5 KO
100
200
300
- +Flagellin: - +WT T5KO
Seru
m K
C [n
g/m
l]
4
8
12
- +Flagellin: - +
Seru
m G
-CSF
[ng/
ml]
WT T5KO
TLR5KO=T5KO
Goal: Does loss of TLR5 reduce inflammation in models of IBD?
Prediction: Colitis driven by “immune dysregulation” might be reduced by loss of TLR5
WTWT T5KOT5KO0.00.0
0.50.5
1.01.0
1.51.5
2.02.0
2.52.5
40%40%
WTWT T5KOT5KO00
11
22
33
44
28%28%
WTWT T5KOT5KO0.00.0
0.30.3
0.60.6
0.90.9
1.21.2
1.51.5
27%27%
Col
on w
eigh
t [%
bod
y m
ass]
Col
on w
eigh
t [%
bod
y m
ass]
Cec
um w
eigh
t [%
bod
y m
ass]
Cec
um w
eigh
t [%
bod
y m
ass]
Sple
en w
eigh
t [%
bod
y m
ass]
Sple
en w
eigh
t [%
bod
y m
ass]
T5KO mice develop spontaneous colitisT5KO mice develop spontaneous colitisT5KOT5KO
(10%)(10%)52/50052/500WT WT
500/500500/500
Vijay-Kumar et al. J Clin Invest 2007
WTWT T5KOT5KO11
100100
1000010000
SAA
[SA
A [ µµ
g/m
l]g/
ml]
WTWT T5KOT5KO--LL T5KOT5KO--HH00
11
22
33
Col
on [g
%bo
dy w
t]C
olon
[g%
body
wt]
**
WTWT T5KOT5KO--LL T5KOT5KO--HH00
11
22
33
Cec
um [%
bod
y w
t]C
ecum
[% b
ody
wt]
**
WTWT T5KOT5KO--LL T5KOT5KO--HH0.000.00
0.250.25
0.500.50
0.750.75
Sple
en [%
bod
y w
t]Sp
leen
[% b
ody
wt]
****
WTWT T5KOT5KO--LL T5KOT5KO--HH00
1010
2020
3030
4040
Col
on M
PO [U
/g ti
ssue
]C
olon
MPO
[U/g
tiss
ue]
**T5KOT5KO--HH
T5KOT5KO--LL
Serum Amyloid A marks severity of T5KO Serum Amyloid A marks severity of T5KO colitiscolitis
Rectal Prolapse
WTWT T5KOT5KO
101055
Feca
l CFU
/gFe
cal C
FU/g
101033
101099
101077
WTWT T5KOT5KO101022
Col
onic
Col
onic
-- adh
eren
t CFU
/gad
here
nt C
FU/g
101033
101044
101055
WTWT T5KOT5KO11
1010
100100
10001000
Live
r CFU
/g ti
ssue
Live
r CFU
/g ti
ssue
WTWT T5KOT5KO11
1010
100100
10001000
Sple
en C
FU/g
tiss
ueSp
leen
CFU
/g ti
ssue
T5KO mice have an increased bacterial burdenT5KO mice have an increased bacterial burden
T5KO mice have an increased bacterial invasionT5KO mice have an increased bacterial invasion
WT T5KO
UntreatedT5KO-H
+ Streptomycin10
100
1000
SAA
[µg/
ml]
*
Antibiotic treatment ameliorates T5KO colitis
Untreated + StrepWT
T5KO
Transfer embryo
“Rederived offspring”Acquires microbiota of birth (recipient)
mother but not genetic mother
Embryo transfer changes environment but notgenetics
Rectal Prolapse: 0%
*
WT T5KO0
5
10
15
20
25
Cec
um M
PO [U
/g ti
ssue
]
*
WT T5KO0
100
200
300
Seru
m L
ipoc
alin
2 [n
g/m
l]
**
WT T5KO0.0
0.1
0.2
0.3
0.4
Sple
en [%
Bod
y W
eigh
t]
***
WT T5KO0.00
0.25
0.50
0.75
1.00
Col
on [%
Bod
y W
eigh
t]
Rederivation via embryonic transplant amelioratescolitis in T5KO mice
Potential mechanism of T5KO colitis
Persistent Inflammation ofthe Gut and Colitis
Decreased Epithelial Barrier Function
Rectal Prolapse
Dissemination of Bacteria
Anemia, Splenomegaly, Leukocytosis
Acute Phase Response(SAA, Lipocalin)
Loss of TLR5 Function
Failure to Manage Microbiota
Increased Activation of Hemopoietic TLRs
Increased Expression of TH1Cytokines(IL-23, IL-12, IFNγ,TNFα)
II. TLR5 deficiency results in metabolic syndrome
I. TLR5 deficient mice develop spontaneous colitis
WT T5KO15.0
17.5
20.0
22.5
25.0
Bod
y W
eigh
t [g]
*(20-22%)
T5KO mice are overweight
Regular DietMales and Females
Before and after Rederivation
Vijay-Kumar et al (2010) Science
WT Leptin-KO(Ob/Ob)
WT TLR5-KO
I’m not obese.I’m robust!
(12 week old ) (18 week old)
Michel Angelo’s David ….. David after short stay in USA…..
National Health and Nutrition Examination StudyBy 2030, 86% US adults will be overweight/Obese
9.1% of Total Medical Expenditure in 2010 (168 Billions)
Wang et al Obesity 2008
WT T5KO
1. Body Weight2. Abdominal Fat Pad
II. Blood Chemistry1. 15h Fasting Blood Glucose2. Serum Triglycerides3. Serum Cholesterol4. Serum Insulin5. Serum Leptin6. Serum NPY
III. Functional Tests1. Glucose Tolerance Test2. Insulin Sensitivity Test3. Food Intake4. Stool Output5. Blood Pressure
IV. Cecal Microbiota1. 16s rRNA Sequencing
I. Gross Phenotype
Metabolic examination of T5KO mice
T5KO mice develop obesity
4 6 8 10 12 14 16 18 20
20
25
30
35
W eek
Bod
y W
eigh
t (g)
4 6 8 10 12 14 16 18 20
15
20
25
W eek
Bod
y W
eigh
t (g)
WT T5KO Males Females
WT
T5KO
WT
T5KO
WT T5KO50
75
100
125
150
175
Seru
m C
hole
ster
ol [m
g/dL
]
60
80
100
120
140
160
Bloo
d Pr
essu
re [m
mHg
]WT T5KO WT T5KOsystole diastole
**
**
WT T5KO
50
60
70
80
Seru
m T
rigly
ceri
des
[mg/
dL]
WT T5KO
0.25
0.50
0.75
Fat P
ad [g
]
MalesWT T5KO WT T5KO
WT T5KO
0.1
0.2
0.3
Females**
T5KO exhibit hyperglycemia/insulin resistance
0.4
0.8
1.2
1.6
Seru
m In
sulin
[ng/
ml]
Glucose: - + - +WT T5KO
*
*
WT T5KO60
85
110
135
15h
Fast
ing
Blo
od G
luco
se [m
g/dL
]
*
WT T5KO
0.25
0.50
0.75
1.00
Seru
m In
sulin
[ng/
mL]
0 30 60 90 12050
60
70
80
90
100
Time [min]
Blo
od G
luco
se [%
sta
rtin
g]T5KO
WT
Glucose Tolerance
0 30 60 90 120
100
200
300
400
WTKO
Time [mins]
Blo
od G
luco
se [m
g/dL
]
+ Insulin
WT T5KO
Islets
Insulinstain
WT T5KO
I. DietHigh Fat Diet for 8 weeks
II. Calorie IntakeCalorie Restriction (12 weeks)
III Microbiota AblationAmpicillin and Neomycin (12 weeks)
VI. Gnotobiotic T5KO miceStudying MetS in germ-free T5KO in Comparison to germ-free WT mice
V. Microbiota TransferTransfer Cecal Microbiota to Germfree Mice
Interventional examination of T5KO mice
High fat diet aggravates metabolic syndrome in T5KO mice
WT T5KO
1
2
3
4
Fat P
ad [g
]
WT T5KO
20
25
Bod
y W
eigh
t [g]
*
WT T5KO
100
125
150
15h
Fast
ing
Glu
cose
[mg/
dL]
**
WT T5KO
0.25
0.50
0.75
Seru
m In
sulin
[ng/
mL]
*
WT T5KO
30
40
50
60
Seru
m T
rigly
cerid
es [m
g/dL
]
*
WT T5KO100
150
200
250
300
Seru
m C
hole
ster
ol [m
g/dL
]
*
WT T5KO0
10
20
30
40
Seru
m L
eptin
[ng/
mL]
*
WT T5KO
Pancreas(Insulitis)
Liver(Steatosis)
T5KO mice exhibit hyperphagia
WT T5KO2.5
3.0
3.5
Food
Inta
ke [g
/mou
se/d
ay]
*
WT T5KO0
100
200
300
Wet
sto
ol o
utpu
t [m
g/h] *
*
WT T5KO0
25
50
75
100
125
Dry
Sto
ol o
utpu
t [m
g/h]
Calorie restriction improves metabolicsyndrome in T5KO mice
WT T5KO0.0
0.5
1.0
1.5
Seru
m L
eptin
[ng/
mL]
WT T5KO
25
50
75
100
15h
Fast
ing
Glu
cose
[mg/
dL]
WT
0.10
0.15
0.20
Fat P
ad [g
]WTT5KO T5KO
Males Females
WT T5KO0
50
100
Seru
m C
hole
ster
ol [m
g/dL
]
WTT5KO
0 20 40 600
25
50
75
Days
% W
eigh
t Gai
n
0 30 60 90 120
40
60
80
100
WT
T5KO
Time [min]B
lood
Glu
cose
[% s
tart
ing
valu
e]
+ Insulin
T5KO colons have elevated innate immune T5KO colons have elevated innate immune gene expressiongene expression
Pattern Recognition ReceptorsTLR4CD14LBP
Lumican
AntimicrobialsRegIIIγ
SLPIiNOSIDO
LipocalinsDefensins
FcRn
CytokinesS100A9IL-1βTNFαIL-10IL-12CSF-1
Acute phase proteinsHaptoglobin
SAAHepcidinLipocalin
Antioxidant enzymesGPxGSTGR
SOD
Stress proteinsHSP70HIF1αHSP12HSP1
ROS enzymesNOX1NOX3NOA-1NOO-1MPO
Adhesion moleculesCEACAM-10CEACAM-13
P-SelectinVCAM-1ICAM-2
Vijay-Kumar et al J. Clin. Invest. 2007
TLR5KO
Vijay-Kumar et al Infec. Immun. 2008
Does loss of TLR5 alter composition of the gut microbiota?
WT T5KO
Extract cecal contents (n= 5 mice/genotype)
Subject to 454 sequencing of 16S rRNA(approx 2500 sequences/mouse)
UNIFRAC
Survey says…
Ruth LeyCornell Univ
T5KO mice exhibit taxonomical alterations in the gut microbiota
No major shift in Bacteroidetes and Firmicutes
Enriched Phylotypes (in KO):Bacteroidetes:Firmicutes:Proteobacteria:
Depleted Phylotypes (in KO):Bacteroidetes:Firmicutes:Proteobacteria:
3518161
7751242
WT
T5KO
Untreated + Antibiotics
WT
T5KO
WT T5KOAntibiotics: - + - +
**
*
0
1
2
3
4
Cec
al B
acte
ria/g
[x10
12]
Antibiotics Normalizes Gut Bacteria in WT and in T5KO mice
Ampicllin+Neomycin
Antibiotics prevent metabolic syndrome in T5KO mice
WT T5KO
2.5
3.0
3.5
4.0
4.5
5.0Fo
od In
take
[g/m
ouse
/day
]
WT T5KO
0.3
0.4
0.5
Fat P
ad [g
]
15h
Fast
ing
Glu
cose
[ mg/
dL]
WT T5KO
70
75
80
85
90
WT T5KOWeaning Post-Abx
WT T5KO WT T5KOWeaning Post-Abx
50
100Se
rum
Cho
lest
erol
[mg/
dL]
150
WT T5KO0.0
0.1
0.2
0.3
0.4
Sple
en [g
/100
g B
W]
WT T5KO0.0
0.2
0.4
0.6
0.8
Col
on [g
/100
g B
W]
WT T5KO0
10
20
30
Bod
y W
eigh
t [g]
WT T5KO0.0
0.2
0.4
0.6
0.8
1.0
Fat P
ad [g
/100
g B
W]
Germ free T5KO mice do not exhibit metabolicsyndrome
T5KO microbiota is necessary and sufficient to transfer metabolic syndrome to WT germfree mice
WT donor T5KO donor60
80
100
120
WT donor T5KO donor
4.0
4.5
5.0
5.5
0 20 40 600
50
100
Days post transplant
% W
eigh
t Gai
n
WT donor T5KO donor
0.50
0.75
1.00
Food
Inta
ke[g
/mou
se/d
ay]
Seru
m In
sulin
[ng/
ml]
15h
Fast
ing
Glu
cose
[m
g/dL
]
***
WT donor T5KO donor0 50 100 150 200
50
75
100
Time [min]
Blo
od G
luco
se [%
sta
rtin
g va
lue]
T5KO
WT
0
50
100
TNFα IL-1β
*
WT T5KO
Col
on C
ytok
ine
[pg/
mL
] 150
0
50
100
*
WT T5KO
+ Insulin
SummaryMetabolic Syndrome in T5KO mice is
1. Hyperphagia
2. Aggravated by High fat diet
3. Treatable by Calorie Restriction and Antibiotics
4. Transferable to Germ-free mice by Cecal Microbota
5. GF-T5KO mice do not suffer from Metabolic Syndrome
Trait Penetrance (%)
TLR5-stop (null) Genotype
WT(n=13,000)
Heterozygous(n=1400)
Homozygous(n=52)
Hyperglycemia (FG>110)
Large waist (>102 or 88 cm for M or F)
P-value WT vs. Homozyg.
High TG (>149)
Hypertension (>130 or 85 for S or D)
Low HDL (< 41 or 51 for M or F)
32.4 34.6 42.6
35.4 36.1 44.9
36.1 38.1 38.0
23.4 24.9 34.6
76.2 78.7 84.0
0.39
0.08
0.09
0.02
0.07
Relevance in humans?
About 1 in 250 humans lacks TLR5 function due to the“TLR5-stop” SNP
(T.R. Hawn, Aderem and colleagues) Karen Mohlke (UNC)
Proposed Mechanism
Loss of TLR5
Altered host-microbiota interactions
HyperglycemiaHyperinsulinemia
Hyperlipidemia, HypertensionDiabesity
Insulin resistance Hyperphagia
IL-1β and other pro-inflammatory cytokines(Low-grade chronic or sub-clinical inflammation)
Acknowledgements
Andrew GewirtzJesse AitkenFred CarvalhoDan MooreBenyue ZhangSindhu Srinivasan
Ifor WilliamsShanthi SitaramanShanthi SrinivasanSimon Mwangi
Host Defense, Osaka, JPSatoshi Uematsu
Shizuo Akira
Emory University
Cornell UniversityRuth Ley
Tyler Cullendar
U. ColoradoRob Knight
U. North CarolinaKaren Mohlke