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