Immunology, Faculty of Medicine

University of Bari, Bari (Italy)

Proprietà benefiche di latte, vino e

olio extra vergine di oliva

Professor Emilio Jirillo

mucosa-associated lymphoid tissues (MALT)

➢ gut-associated lymphoid tissues (GALT)

➢ nasal-associated lymphoid tissues (NALT)

➢ bronchus-associated lymphoid tissues (BALT)

➢ genitourinary-associated lymphoid tissues (GUALT)

➢ Peyer’s patch (PP)

➢ isolated lymphoid follicle (ILF)

➢ intraepithelial lymphocyte (IEL)

➢ lamina propria (LP)

basketball court(about 400 m2)

M

T cell

B cell

DC

plasma cell

Peyer’s patch

M

secretory IgA

lamina propria

IEL

MLN

gut-associated lymphoid tissues (GALT)

M

TGF-

IL-4, IL-5

T cell

B cell

DC

sIgA+ B cell

PP

M

IL-5, IL-6

plasma cell

pIgR

secretory IgA

lamina propria

inductive tissue effector tissue

Production of secretory IgAby the gut immune system

Milk effects on the immune system

Effects of red grape polyphenols on the immune

system in experimental models and humans

General structure of flavonoids

HO

OH

OHResveratrol

Resveratrol3,4’,5 –trihidroxy-trans-stilbene

fitoalexin

Non flavonoids compoundO

O

A

B

C

1

2

34

5

6

7

81'

2'

3'

4'

5'

6'

C6-C3-C6

Ring skeletons of flavonoids

O

OFlavanone

O

O

Isoflavone

O

OFlavonol

OH

O

OH

+

Anthocyanidin

O

OFlavanonol

OH

O

OH

O

OHProanthocyanidin

O

Flavanol OH

Polymer form of Flavanols

Flavone

O

O

Endothelial cells

NO EDHFET-1 MCP-1I-CAM V-CAM

Proliferation

Migration

HO

OH

OH

Polyphenols

M

Platelet

Oxidized LDL

Foam cell formation

Aggregation

Adhesion

TLR4

LPS

MD

-2

MD

-2

LPS

p65

IB

p50

IB

Proteasome

NF-B

IKK-

MEK1/2 MKK3

MKK7

JNK

p38

MA

L

IRAK4IRAK2

IRAK1

TRAF6

IKK

-

IKK

-

UBC13

UBV1A

My

D8

8

LBP

sCD14

LPS

TAB1

TAB2 TAK1

cytokines,NO

polyphenols

Fermented Grape Marc (FGM) and

Intestinal Immunity

H2O ethanol H2O ethanol

Koshu Negroamaro

flavonol: yellow

catechin, epicatechin: green

flavonol: yellow

++

catechin, epicatechin: green

catechin, epicatechin: green

catechin, epicatechin: green

THE POLYPHENOLS FAMILY

Koshu Negroamaro Tannat

Materials for our experiments

(Japan) (Italy) (Italy)

The manufacturing process of FGM

Grape marcSupermasscolloider

(Ultra-fine friction grinder)Grape marc : H2O = 1 : 1

fermentationfor 4 days

freeze-drying

sterilization at121 °C, 15 min

add. 5% (v/v)incubate for 1 day

sample (FGM)

1% lactic acidbacteria media

(Lactobacillus plantarum)

Inflammatory bowel disease (IBD)

ulcerative colitis Crohn’s disease

inflammatory area colon gastrointestinal tract

damage shallow intestinal wall deep into the layers ofintestinal wall

cytokineresponse Th2 dominant Th1 dominant

0 1 2 3 4 5 6 7 days

FGM (10, 30, or 100 mg/kg in water, p.o.)

BALB/c mice

body weight

colon length

5% dextran sodium sulfate (DSS)

TNF- or IL-1production in homogenized colon

Induction of colitis

Normal group

Control group

FGM-treatedgroup

5% DSS p.o.

-

+

+

water

water

each ofFGMs

20

40

60

80

100

120

0perc

ent

age o

f bod

y w

eig

ht

loss

(%

)

Con

trol

fermentation (100 mg/kg)

Negr

oam

aro

Kos

hu

Tan

nat

Kos

hu

Effect of FGM on body weight loss

0

20

40

60

80

100

perc

ent

age o

f sh

orte

ning

of

colo

rect

um (

%)

*

Con

trol

fermentation (100 mg/kg)

Negr

oam

aro

Kos

hu

Tan

nat

Kos

hu

Effect of FGM on length of colon

Normal

Control

10

30

100

fermentedKoshu

(mg/kg)

Comparison of length of colon in FGM (Koshu)-treated mice

0

200

400

600

800

1000

1200

1400

0

500

1000

1500

2000

2500

IL-1βTNF-α

pg/m

l

pg/m

l* * *

**

Nor

mal

Con

trol 10 30 100

fermented Koshu (mg/kg) Nor

mal

Con

trol 10 30 100

fermented Koshu (mg/kg)

Effect of FGM (Koshu) on inflammatory cytokines in homogenized colon

Fermented Grape Marc and effects on

human PBMCs

Evaluation of cytokine release from human PBMCs stimulated by N-FGM and K-FGM

N-FGM/ water N-FGM/ethanol K-FGM/water

K-FGM/ethanol

IL-12(monocytes)

u.n. 9% 17% u.n.

IL-10 (lymphocytes)

u.n. 20% u.n. 6%

IL-10 (monocytes)

6% u.n. 7% u.n.

TNF-(monocytes)

8% u.n. u.n. 7%

% increase vs untreated cellsu.n. undetectable

Intracellular content of cytokines in human PBMCs

FoxP3

Ab anti-CD4

Ab anti-FoxP3

Ab anti-CD25CD25+

CD4+

Intranuclear staining of FoxP3 in Treg cells

unstimulated Positive control (PMA) FGM-stimulated cells

N/wNegroamaro/water

N/etNegroamaro/ethanol

K/wKoshu/water

K/etKoshu/ethanol

FoxP3 n.s. n.s.

FoxP3 expression

Percent induction of Treg cells by N-FGM and K-FGM

GrB release by human PBMCs stimulated with N-FGM and K-FGM

Detection of Granzyme B in supernatant of lymphocytes culture

Effects of FGM on granulocytes and monocytes

Effects of Extra Virgin Olive Oil on the Immune-Mediated

Inflammatory Responses: Potential Clinical Applications

In vitro polyphenol effects on the

immune system from obese people

Determination of absolute numbers of

peripheral blood mononuclear cells (PBMCs)

H u

nstim

CD

3+

Ob u

nstim

CD

3+

H C

D3+

1ug

Ob C

D3+

1 u

g

H C

D3+

3 u

g

Ob C

D3+

3 u

g

H C

D3+

5ug

Ob C

D3+

5 u

g

0

1000000

2.0100 6

3.0100 6

@ @@

@

@p<0.0001 H unstimulated vs Ob unstimulated

@p<0.0001 H 1ug/ml vs Ob 1 ug/ml

@p<0.0001 H 3 ug/ml vs Ob 3 ug/ml

@p<0.0001 H5 ug/ml vs Ob 5 ug/ml

H u

nstim

CD

4+

Ob u

nstim

CD

4+

H C

D4+

1ug

Ob C

D4+

1 u

g

H C

D4+

3ug

Ob C

D4+

3ug

H C

D4+

5ug

Ob C

D4+

5 u

g

0

5.0105

1.0106

1.5106

2.0106

# #

#

# p<0.001 H unstimulated vs Ob unstimulated

# p<0.001 H 1 ug/ml vs Ob 1 ug/ml

# p<0.001 H 3 ug/ml vs Ob 3 ug/ml

* p<0.05 H 5 ug/ml vs Ob 5 ug/ml

*

H u

nstim

CD

8+

Ob u

nstim

CD

8+

H C

D8+

1ug

Ob C

D8+

1 u

g

H C

D8+

3ug

Ob C

D8+

3 u

g

H C

D8+

5ug

Ob C

D8+

5 u

g

0

5100 5

1100 6

2100 6

@ @ @@

@p<0.0001 H unstimulated vs Ob unstimulated

@p<0.0001 H 1ug/ml vs Ob 1 ug/ml

@p<0.0001 H 3 ug/ml vs Ob 3 ug/ml

@p<0.0001 H5 ug/ml vs Ob 5 ug/ml

H u

nstim

CD

19+

Ob u

nstim

CD

19+

H C

D19

+ 1ug

Ob C

D19

+ 1 u

g

H C

D19

+ 3ug

Ob C

D19

+ 3 u

g

H C

D19

+ 5ug

Ob C

D19

+ 5 u

g

0

510 0 5

110 0 6

210 0 6

#

# p<0.001 H unstimulated vs Ob unstimulated

@ p<0.0001 H 1 ug/ml vs Ob 1ug/ml

*p<0.05 H 3 ug/ml vs Ob 3 ug/ml

@

*

H u

nstim

CD

16C

D56

+

On u

nstim

CD

16+56

+

H C

D16

CD

56+ 1

ug

Ob C

D16

+CD

56+ 1

ug

H C

D16

CD

56+ 3

ug

Ob C

D16

+CD

56+ 3

ug

H C

D16

CD

56+ 5

ug

Ob C

D16

+CD

56+ 5

ug

0

5100 5

1100 6

2100 6

@ @ # #

@p<0.0001 H unstimulated vs Ob unstimulated

@p<0.0001 H 1 ug/ml vs Ob 1 ug/ml

#p<0.001 H 3 ug/ml vs Ob 3 ug/ml

#p<0.001 H 5 ug/ml vs Ob 5 ug/ml

Polyphenols do not influence the

increased absolute numbers of obese

PBMCs

Effects of polyphenols on Th1 related-cytokines

IL-2 release

IFN- release

In vitro treatment with polyphenols did not affect the

release of both IL-2 and IFN-

Effects of polyphenols on Th2 related-cytokines

IL-4 release

In vitro treatment with polyphenols did not affect the release of IL-4

The enhanced release of Th1 and Th2-related

cytokines in the presence of PMA suggests a

condition of immune hyperactivation in obese

people as also indicated by the increase in

PBMC absolute numbers

Effects of polyphenols on Treg/Th21 axis

The inflammatory /anti-inflammatory

pathway

T regulatory cells

Production of IL-10, the anti-

inflammatory pathway

Inhibition of Th17 cells

(IL-17A-F), the

inflammatory pathway

IL-21 operates as

an inductor of Th17

Spontaneous release of IL-17 in obese people was absent, thus

depending on both IL-10 production and IL-21-induced suppression

by polyphenols.

Spontaneous release ofIL-10 is moderatelyincreased by polyphenols

Release of IL-21, an inducer

of Th17 cells, was abrogated

in the presence of polyphenols

while they significantly

enhanced production of this

cytokine by normal PBMC

In vitro effects of polyphenols on pro-inflammatory

cytokine release

Reduced release of IL-1 and IL-6 from obese PBMCs by polyphenols in comparison to

the normal counterpart reflects the anti-inflammatory pathway above described