intestinal microflora and immunoregulation

Falk Workshop. Mechanisms of Intestinal Inflammation, October 9-10, 2007. Dresden

Intestinal Microflora and Immunoregulation

Monica Boirivant Istituto Superiore di Sanità

Roma


Microflora-immune cells interactions

T

Ethanol AT 1002


Effect of ir Ethanol 50% administration

80

85

90

95

100

105

110

115

0 2 4 6 8 10 12

Days

wei

ght (

% o

f day

0)

UntreatedEth 50%

* * * *

*= p<0.05


Effect of ir Ethanol 50% administrationIncreased intestinal permeability

0

200

400

600

800

0 1 2 3 4 5 6 7 8

Days

Seru

m F

itc-D

extr

an

(μg/

ml)

* * * *

*p<0.05 vs day 0

Day 1 Day 3

Day 5 Day 7


Effect of ir Ethanol 50% administrationIncreased LPMC production of IFN-γ and IL-10

Unstim

0

200

400

600

800

Untreated Eth50%

IFN

-γ(p

g/m

l)

p=0.02

200

400

600

800

Untreated Eth 50%

αCD3/28

p= 0.03

0.007Unstim

0

200

400

600

800

Untreated Eth50%

IFN

-γ(p

g/m

l)

p=0.02

200

400

600

800

Untreated Eth 50%

αCD3/28

p= 0.03

0.007

0

200

400

600

800

Untreated Eth50%

IFN

-γ(p

g/m

l)

p=0.02

0

200

400

600

800

1000

1200

Untreated Eth 50%

IL-1

0 (p

g/m

l) αCD3/28

p= 0.03

0.007


Effect of ir Ethanol 50% administrationIncreased % of CD25+ and LAP+ cells in CD4+LPC

CD

25+

T ce

lls(%

)

0

2

4

6

8

10

12

14

Untreated Eth 50%

p=0.002

0

2

4

6

8

10

12

14

Untreated Eth 50%

LAP+

T c

ells

(%)

p=0.02


0

10

20

30

40

50

60

70

80

90

CD25+ cells LAP+ cells

Foxp

3+ c

ells

(%) Untreated

Eth 50%


Effect of ir Ethanol 50% pre-administrationProtection from TNBS-colitis

Eth 50%/TNBS

Days

80

85

90

95

100

105

0 1 2 3 4

wei

ght (

% o

f day

0)

TNBS

* * *Eth 50%/TNBS

Days

80

85

90

95

100

105

0 1 2 3 4

wei

ght (

% o

f day

0)

TNBS

* * *Eth 50%/TNBSTNBS

Eth/TNBS TNBS

IFN

-γ (n

g/m

l )

UnstimαCD3/28

0

5

10

15

20

25

30

35 p=0.03

Eth/TNBS TNBS

IFN

-γ (n

g/m

l )

UnstimαCD3/28

0

5

10

15

20

25

30

35 p=0.03

IFN

-γ (n

g/m

l )

UnstimαCD3/28

0

5

10

15

20

25

30

35 p=0.03

*= p<0.05


AT 1002

• Hexapeptide representing the active domain of zonula occludens toxin from Vibrio cholerae

• Acting as mammalian zonulin, causing disassemblingof tight junctions

• AT1002 intraluminal administration is associated withtransient increase of intestinal permeability withoutepitelial damage

Katouzian F et al. J pediatr Gastroenterol Nutr 2005; 40:632Mottlekar NA et al. J Drug Target 2006; 14:321-9


Effect of ir AT 1002 administrationIncreased LPMC production of IFN-γ and IL-10

Increased % of CD25+ and LAP+ cells in CD4+LPC

AT 1002 treated

Untreated20

40

60

80

100

120

140

0Untreated

Unstimα CD3/28

AT 1002 AT 1002U ntreated

IL-10 IFN -γpg/m

l

20

40

60

80

100

120

140

20

40

60

80

100

120

140

0Untreated

Unstimα CD3/28


IL-10 IFN -γpg/m

l

0Untreated

Unstimα CD3/28Unstimα CD3/28


IL-10IL-10 IFN -γpg/m

l%

U n t r e a t e d

0

1

2

3

4

5

6

7

C D 2 5 L A P

A T 1 0 0 2

p = 0 .0 2

%U n t r e a t e d

0

1

2

3

4

5

6

7

C D 2 5 L A P

A T 1 0 0 2

p = 0 .0 2

U n t r e a t e d

0

1

2

3

4

5

6

7

C D 2 5 L A P

A T 1 0 0 2

p = 0 .0 2


Effect of ir AT1002 pre-administrationProtection from TNBS-colitis

70

75

80

85

90

95

100

105

0 1 2 3 4

wei

ght (

% o

f day

0)

AT1002/

TNBS

* * * *

0 1 2 3 4 5

AT1002/TNBS

TNBS

ColitisscoreDays

TNBS

70

75

80

85

90

95

100

105

0 1 2 3 4

wei

ght (

% o

f day

0)

AT1002/AT1002/

TNBS

* * * *

0 1 2 3 4 5

AT1002/TNBS

TNBS

ColitisscoreDays

TNBS

*= p<0.05


Summary I

• Specific induction of increased mucosal permeability is associated with an increasedproduction of IFN-γ and IL-10 as well as withan increase in the number of CD4+T cellsexpressing CD25 and/or LAP

• This condition is associated with resistance toinduction of TNBS colitis


Effect of ir Ethanol 50% pre-administrationProtection from TNBS-colitis is dependent on LAP+Tcells

70

75

80

85

90

95

100

105

0 1 2 3 4 5Days

wei

ght(

% o

f day

0)

Recipients of:

LPMC fromethanol treatedmice (A)

LPMC fromethanol treated

LAP depleted

Mice (B)

LPMC fromTNBStreated mice (C)

TNBS(D)

Recipientsof LAP+depletedLPMC

0102030405060708090

100

Recipientsof LPMC

TNBSIF

N-γ (

ng/m

l)

UnstimαCD3/28

Recipientsof LAP+depletedLPMC

0102030405060708090

100

Recipientsof LPMC

TNBSIF

N-γ (

ng/m

l)

UnstimαCD3/28

0102030405060708090

100

Recipientsof LPMC

TNBSIF

N-γ (

ng/m

l)

UnstimαCD3/28


100 101 102 103 104100

101

102

103

104

6.7

100 101 102 103 104100

101

102

103

104

9.3

100 101 102 103 104100

101

102

103

104

9.31

100 101 102 103 104100

101

102

103

104

7.27

100 101 102 103 104100

101

102

103

104

24.8

100 101 102 103 104100

101

102

103

104

12.3

Control ETOH ETOH(LAP depleted)

CD4

LAP

Foxp

3

100 101 102 103 104100

101

102

103

104

0.75

100 101 102 103 104100

101

102

103

104

0.28

Goa

tIgG

Rat

IgG

CD4


CD4+LAP+ cells expansion is dependent on the presenceof an intact bacterial flora

20

0

5

10

15

25

30

CD25+ LAP+

%

Antibiotics/Eth50%

Eth 50%

Untreated

70

75

80

85

90

95

100

105

0 2 4

Days

Eth 50%

Antibiotics/Eth 50%

531

wei

ght (

% o

f day

0)

20

0

5

10

15

25

30

CD25+ LAP+

%

Antibiotics/Eth50%

Eth 50%

Untreated

Antibiotics/Eth50%

Eth 50%

UntreatedUntreated

70

75

80

85

90

95

100

105

0 2 4

Days

Eth 50%

Antibiotics/Eth 50%

Eth 50%

Antibiotics/Eth 50%

531

wei

ght (

% o

f day

0)

Ampicillin:1g/L

Vancomycin: 500 mg/l 4 weeks

Neomycin: 1g/L

Metronidazole: 1g/L


Summary II

• CD4+LAP+ cells are responsible for the protection from TNBS colitis

• CD4+LAP+ expansion is dependent on the presence of an intact bacterial flora


Plasmacytoid dendritic cells do not play a role in the expansion of regulatory cells causing increased resistance to TNBS colitis

75

80

85

90

95

100

105

0 1 2 3 4

Days

wei

ght (

% o

f day

0)

120G8 /Eth /TNBSEth/ TNBS120G8/TNBSTNBS

75

80

85

90

95

100

105

0 1 2 3 4

wei

ght (

% o

f day

0)

αGr-1/Eth/TNBS

αGr-1/TNBS

TNBS

Eth/TNBS


Myeloid dendritic cells induce IL-10 dependent expansionof CD4+LAP+T cells

0

2

4

6

8

10

12

14

16

LP CD4+ cellsuntreated miceCD11c+ cellsuntreated mice

anti -IL-10R

CD11c+ cellsethanol –treated mice

LAP+

cells

%

+ + + + +

_

_ _+ __ +_

_

+ +

_ + _ +

0

2

4

6

8

10

12

14

16


anti -IL-10R


LAP+

cells

%

+ + + + +

_

_ _+ __ +_

_

+ +

_ + _ +

0

2

4

6

8

10

12

14

16

LP CD4+ cellsuntreated miceCD11c+ cellsuntreated miceCD11c+ cellsuntreated mice

anti -IL-10R

CD11c+ cellsethanol –treated miceCD11c+ cellsethanol –treated mice

LAP+

cells

%

+ + + + +

_

_ _+ __ +_

_

+ +

_ + _ +


TLR2 signaling is critical for the generation of LAP+ Regulatory cells

siRNA TLR2/Eth 50%

Eth 50%

siRNA Control /Eth 50%

80859095

100105110115

0 1 2 3 4 5 6 7 8Days

Wei

ght (

% o

f day

0)

siRNA TLR2/Eth 50%siRNA TLR2/Eth 50%

Eth 50%Eth 50%

siRNA Control /Eth 50%siRNA Control /Eth 50%

80859095

100105110115

0 1 2 3 4 5 6 7 8Days

Wei

ght (

% o

f day

0)

p=0.01

pg/m

l

Eth50%

IFN-γ IL-10-0

100200300400500600700800900

100011001200

LAP+

cells

(%)

Eth5

0%

p= 0.005

012345

678

p=0.01

siC

tr/E

th50

%

siTL

R2/

Eth5

0%

siCtr/Eth50%siTLR2/Eth50%


TLR2 signaling is critical for the generation of LAP+ regulatory cells

siRNA TLR2/Eth 50%/TNBS

Eth 50%/ TNBS (A)siRNA Control /Eth 50%/ TNBS (B)

TNBS(D)

75

80

85

90

95

100

105

0 1 2 3 4

Days

wei

ght(

% o

f day

0)

siRNA TLR2/Eth 50%/TNBS (C)

Eth 50%/ TNBS (A)Eth 50%/ TNBS (A)siRNA Control /Eth 50%/ TNBS (B)siRNA Control /Eth 50%/ TNBS (B)

TNBS(D)

75

80

85

90

95

100

105

0 1 2 3 4

Days

wei

ght(

% o

f day

0)


Summary III

• Myeloid dendritic cells induce an IL-10 dependent expansion of CD4+LAP+T cells

• TLR2 signaling is critical for the generation of LAP+ regulatory cells


Conclusions

• Transient loss of epithelial cell barrierfunction increases the resistance of the mucosa to inflammation via the induction of regulatory T cells

• Limited exposure of the mucosal immune system to the microflora might represent animportant mechanism of tolerance inductionand gut homeostasis


• Antonello Amendola• Alessia Butera• Monica Boirivant

• Ivan Fuss• Warren Strober

• Immune-mediated Diseases Section. Department of Infectious , Parasitic and Immune-mediated Diseases. Istituto Superiore di Sanità. Roma. Italy

• Mucosal Immunity Section. Laboratory of host defences. NIAID. NIH. USA


IL-10 production of LPMC before and after LAP depletion

0

20

40

60

80

100

120

140

160

IL-10 TGF-b

pg/m

Untreated

Eth 50%

Eth 50% LAPdepleted


0

50

100

150

200

250

IL-1

0 (p

g/m

l)


anti-IL-10R


+ + + + +

__ _

+ __ +_

_+ +

_ + _ +


0

100

200

300

400

500

600

700

800

900IL

-12

p70

(pg/

ml)


anti-IL-10R


+ + + + +

__ _

+ __ +_

_+ +

_ + _ +

NA

NA : Notavailable


200

400

600

800

1000

1200IL

-10

(pg/

ml)

200

400

600

800

1000

1200IL

-10

(pg/

ml)


anti-IL-10R


+ + + + +

__ _

+ __ +_

_+ +

_ + _ +

ND

ND: not detectable



anti-IL-10R


+ + + + +

__ _

+ __ +_

_+ +

_ + _ +

5

10

15

20

25

30

IL-1

2 p7

0 (p

g/m

l)

ND ND ND0

ND: not detectable


0

50

100

150

200

250

300

350

400

450

500

pg/m

l

IL-12IL-10

IL-12 0 15.58IL-10 8.5 475

CD11c+cells untreated CD11c+cells Eth 50%ND

ND: not detectable


Microflora-immune cells interactions

B MФDC

T

intestinal microflora and immunoregulation

Documents