Prebiotics strengthen

the immune system

Paul Vos, PhD

Danone Research -

Centre for Specialised Nutrition

Wageningen

19-6-2009

Outline

Immune system

Immune regulation

Immune disorders

Natural imbalance

Oligosaccharides: immuno-nutrition breakthrough

In vitro results

In vivo preclinical results

Results from clinical trials

3

Major tasks of the immune system

Defence against pathogens: bacteria, viruses, parasites, fungi

Assistance in anti-cancer responses

Removal of foreign / non-self compounds

Inhibition self-reactive / auto-reactive responsiveness

Inhibition “over-activity/allergy”

YY

Granulocyte

Macrophage / Dendritic cell

Monocyte

T Lymphocyte

(Th1, Th2, Th3, Tr, ...)

B Lymphocyte / Antibodies

Natural Killer Cell

Epithelial cell

Bone marrow

Lymph nodes

Spleen

Thymus

Organs Cells

60 – 70 % of immune cells in the gastro- intestinal tract !!

Immune system

Non-specific first line defence: skin, lungs, intestine,

urinary system (physical/chemical barrier, low pH,

enzymes, mucus, ...)

Non-specific (innate) immunity:

(Macrophages, granulocytes, natural killer cells,..)

Adaptive immunity:

(T Cells, B Cells, memory, antibodies, vaccination)

Immune system

Humoral immunity:

• Antibodies

Cellular immunity:

• Immune-regulation

• Cytotoxicity

Adaptive immunity

T

TT

TT

T

IL-2

T

T

ExpansionActivation

Virus/bacteria/allergen

DC

B

Antibody Production

YYYYYYYY

The adaptive immune response

DCs

Th0Th1/Th17 Th2

APC

Immune regulation

Th0Th1/Th17 Th2

APC

Immune regulationTh1/Th2 paradigm

Allergy

Antibody-mediated autoimmunity

Resistance to intracellular

infections

Anti-cancer

Delayed type

hypersensitivity reactions

Resistance to

extracellular infections

Tolerance

T-cell mediated autoimmunity

Graft rejection

Hallmark signaling molecules / antibody class:

Th1: IFN- , IL-12

Th2: IL-4, IL-13 / IgE antibodies

Tr/Th3: IL-10, TGF- 1 / IgG4 antibodies

Effector cellsActivation

Regulatory cellsInhibition, tolerance

Treg

Th3

Th1/Th17 Th2

APC / DC

Immune regulationThe extended Th1/Th2 paradigm

Immune regulation in the gut

Hyper immune- responsiveness:

Allergy

Autoimmunity

Chronic inflammatory diseases

Hypo immune- responsiveness:

Infections

Tumors/metastasis

Immune disorders (1)

Th1 , Th2 , Th1/Th2

Th1

Th2 (type I allergy)

Th2

Th2

Th1

Th1 ?

Th1

Th1

Th1 , Th2

Immune disorders (2)

HIV

COPD

Allergies

Asthma

Atopic eczema

Coeliac disease

Cystic Fibroses

Cancer

Elderly

Infants

Natural imbalance

Pregnancy and neonates

Th2-directed immune system: cytokines downregulate cellular

immunity (Th1) no rejection of fetus and or mother tissue

After birth

Development of their own “healthy” Th1/Th2 balanced immune

system

Th 2

Normal

PregnanciesFailure

CD 69+

IL 2

TNF α

TNF δ

CD 94+

IL 4

IL 5

IL 10

Th 2Th 1

Normal

Pregnancies

• Genes

• Hygiene

• Drugs

• Diets

• Stress

• Hormones

years0 1 2 5 10 40 50 60 70 80 90

Th 1 Th 2

Th1 and Th2 activity as a function of age

Allergy

Natural imbalance

years0 1 2 5 10 40 50 60 70 80 90

Th 1 Th 2

Stimulate development of Th1/Th2 balanced

immune system during the first year of life

Field et al J Nutr 2005; 135: 1-4

Compounds with immunological properties in human milk Anti-microbial compounds Immune development Immunoglobiuines: sIgA, SIgG, compounds SIgM Macrophages Lactoferrin, lactoferrin B and H Neutrophils Lysozyme Lymphocytes Lactoperoxidase Cytokines Nucleotide-hydrolizing Growth factors Antibodies Hormones к-casein and α-lactalbumin Milk peptides Haptocorrin Long-chain polyunsaturated Mucins fatty acids Lactadherin Nucleotides Free secretory component Adhesion molecules Oligosaccharides and pre-

biotics Anti-inflammatory compounds Fatty acids Cytokines: Il-10 and TGFβ Maternal leukocytes and Il-1 receptor anatagonist Cytokines TNFα and Il-6 receptors sCD14 sCD14 Complement and complement Adhesion molecules receptors Long-chain polyunsaturated β-defensin-1 fatty acids Toll-like receptors Hormones and growth factors Bifidus factor Osteoprotegerin Tolerance/priming compounds Long-chain polyunsaturated Cytokines: Il10 and TGFβ fatty acids Anti-idiotypic antibodies Hormones and growth factors

Compounds with immunological

properties in human milk

Composition of Human Milk

53-61 g/l

30-50 g/l

8-10 g/l

Prebiotics

10-12 g/l

Made

of:

Amino Acids Mono Saccharides Fatty acids

Human milk oligosaccharides are not

commercially available

But:

mixture of specific prebiotic oligosaccharides has

been developed

GOS e.g. DP3 FOS e.g. DP10

[Gal( 1-4)Gal( 1-4)Glc [Frc( 2-]8 1)Frc( -1)Glc

90 % GOS: short-chain -Galacto-OligoSaccharides from lactose

10 % FOS: long-chain -Fructo-OligoSaccharides from chicory

Our oligosaccharides concept

Human milk (g/L) Cow's milk (g/L)

Lactose 55-70 40-50

Total oligosaccharides 3.0-15.0 Traces

Facts:

15-25% of total carbohydrates

a very heterogeneous fraction > 100 different structures

variability: genetic control, time of lactation

soluble, non-digestible, fermentable

Hypotheses:

Induce a healthy gut microbiota in the baby

Modulate the developing immune system

Human milk oligosaccharides

Clinical trials

in humans

In vivo

animal

models

In vitro

animal cells

In vitro

human cells

In vitro mechanistic

Translational Research Approach(as recommended by WHO, FDA)

From invited review on oligosaccharides: critical reviews immunology: Paul Vos et al., 2007

Potential mechanisms of immune modulation by orally

applied nog-digestible carbohydrates

Beneficial

bacterial

SCFA, pH

Immune systemPathogen

reduction

Barrier integrity /

Mucoprotection

Bacterial

fermentation

Boehm et al Nutrafoods 2005

Potential mechanisms of immune

modulation by HMOS

HMOS

Composition of the Intestinal Flora

Prebiotics must stimulate one or a limited number of beneficial bacteria, like bifidobacteria or lactobacilli, within the colonic

microbiota

Non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the

colon that can improve host health

Prebiotics must “survive” acidic conditions

Prebiotics must be selectively fermented in

the colon

Prebiotics must evade digestion in the small

intestine

Fuller & Gibson, 1997, Scand J Gastroenterol Suppl (222):32-40

Prebiotic concept

0.8g/100ml

(n=27)

0g/100ml

(n=33)

0.4g/100ml

(n=30)

log

10 o

f C

FU

/g w

et

fae

ce

s (

me

dia

n, IQ

R)

log

10 o

f C

FU

/g w

et

fae

ce

s (

me

dia

n, IQ

R)

0.8g/100ml

(n=27)

0g/100ml

(n=33)

0.4g/100ml

(n=30)

2

3

4

5

6

7

8

9

10

11

12

Reference range (IQR) of breastfed infants (n=15)

10

0

1

2

3

4

5

6

7

8

9

Reference range

(IQR) of breastfed

infants (n=15)

LactobacillusBifidus

Group difference

according to Mann-

Whitney U-test: *

p<0.05 vs. 0.0, # vs. 0.4

**#

* *

Moro et al .J Pediatr Gastroenterol Nutr 2002; 34:291-295

Group difference

according to Mann-

Whitney U-test: *

p<0.05 vs. 0.0, # vs. 0.8

Oligosaccharides promote growth of

healthy bacteria

Beneficial

bacterial

SCFA, pH

Immune systemPathogen

reduction

Barrier integrity /

Mucoprotection

Bacterial

fermentation

Boehm et al Nutrafoods 2005

Potential mechanisms of immune

modulation by HMOS

HMOS

Improve gut barrier/immunomodulation

Inflammation

induced tissue

destruction

Intestinal

Immune Cell activity

Epithelial

Function and

Regeneration

Permeability

Mesenchym

Mucus

SCFA, SIgA

Short chain fatty acids stimulate mucin-2

production by intestinal epithelial cells

Willemsen et al Gut 2003; 52: 1442-1447

Beneficial

bacterial

SCFA, pH

Immune systemPathogen

reduction

Barrier integrity /

Mucoprotection

Bacterial

fermentation

Boehm et al Nutrafoods 2005

Potential mechanisms of immune

modulation by HMOS

HMOS

Vaccination model

Blood sample

Primary influenza

immunization

Blood sample

Booster influenza

immunization

Study parameters:

• DTH

• Influenza specific antibody titers

• Ex-vivo lymphocyte restimulation

0 7 14 302821-20

Start dietary

intervention

DTH

End

scGOS/lcFOS stimulate Th1-

dependent vaccination response

Vos et al. International Immunopharmacology 2006; 6: 1277-1286

Conclusions so far

GOS/lcFOS can affect systemic immunity (Th1/Th2)

Not all oligosaccharides affect the immune system

What about improved resistance to infections?

What about reduced risk for IgE mediated allergy?

The increase in the prevalence of

asthma, eczema and hay fever

Devereux et al. Nat. Rev. Immunol. 2006;6: 869-874

Sensitisation

Allergen avoidance

Allergen Allergen Provocation

B cellT h 2 cellAntigen-

Presenting (DC) cell

Clinicaleffects

IgE binding

inflammatory cell

IL-4

IL-5

IL-13IgE

Mediators

Mechanism of allergy development

OVA-induced model for experimental

allergic asthma

0 7 35 41

i.p. OVA

Study parameters:

• Airway hyperresponsiveness

• Airway inflammation

• Allergen specific antibody titers

• Ex-vivo lymphocyte restimulation

-14

Start dietary

intervention

38 42

OVA aerosol

challenge

end

Oligosaccharides decrease the manifestation

of allergic asthma

Inflammatory cells in BALAirway hyperresponsiveness

Vos et al., International Immunopharmacology 2007; 7(12):1582-1587

Moro study

Double blind, placebo controlled, randomised prospective study

206 infants with at least a single family history of allergy

gestational age between 37 and 42 weeks (birth weight appropriate for

gestational age)

Feeding regime:

formula feeding within the first 2 weeks of life until the age of 6 months

mixed feeding accepted until the 6th week of life

exclusive formula feeding until the end of 4 months of life

Formulas:

Aptamil HA plus 0.8g GOS/FOS per 100ml (n=104)

Aptamil HA plus 0.8g maltodextrine per 100ml (control; n=102)

Outcome parameters:

primary outcome: atopic dermatitis

Microbiota, IgE and IgG4 in a subpopulation of the cohort

GOS/lcFOS

(n=10/102)Placebo

(n=24/104)

Cu

mu

lati

ve

in

cid

en

ce

of

ato

pic

de

rma

titi

s

0

5

10

15

20

25

30

p<0.03 Fisher‘s exact

Reduction of Atopic Dermatitis and increase in

bifidobacteria in high risk infants at 6 months

Placebo

(n=44)

Bif

ido

ba

cte

ria

co

un

t (l

og

cfu

/g s

too

l)

0

3

6

9

12

15

18

P<0.001 Mann Withney U-test

GOS/lcFOS

(n=50)

10.3

8.7

9.8%

23.1%

Moro et al. 2006, Arch. Dis. Child 91: 814-819

scGOS/lcFOS decrease serum IgE

in High-Risk Infants at 6 Months

**p=0.008

Placebo Gos/lcFos

1

10

100

1000

IgE

(kU

/l)

Hoffen et al. 2009, Allergy 64: 484-487

scGOS/lcFOS decrease CMP IgG1

in High-Risk Infants at 6 Months

P<0.0105

Placebo GOS/lcFOS

0.1

1

10

100

1000

IgG

1 (

AU

/ml)

P<0.0105

Hoffen et al. 2009, Allergy 64: 484-487

Vaccination response is not affected by

scGOS/lcFOS

Placebo Gos/lcFos10

100

1000

10000Ig

G1 (

AU

/ml)

DTP IgG1

Hoffen et al. 2009, Allergy 64: 484-487

Protective effect on infectious episodes

in High-Risk infants at 6 months

Arslanoglu et al, 2007, J .Nutr. 137: 2420-2424

0

10

20

30

40

50

All Infections URTI

infe

cti

ou

s e

pid

so

des

placebo

GOS/lcFOS

p=0.01

trend,

p=0.07

Early dietary intervention with scGOS/lcFOS reduce the

incidence of allergic manifestations during the first 2 years

of life

Arslanoglu et al., 2008, J. of Nutr. 138:1091-1095

Early dietary intervention with a mixture of prebiotic

oligosaccharides reduce the incidence infections during

the first 2 years of life

Arslanoglu et al., 2008, J. of Nutr. 138:1091-1095

Scholtens study

Double blind, placebo controlled, randomized prospective study

215 healthy infants without a family history of atopy

gestational age between 37 and 42 weeks (birth weight appropriate for

gestational age)

Feeding regime:

mixed feeding accepted

Formulas:

Non-hydrolyzed formula plus 0,6g/100 mL scGOS/lcFOS (n=58)

Non-hydrolyzed formula (n=64)

Reference group: full breastfeeding (n=20)

Outcome parameters:

primary outcome: fecal sIgA

scGOS/lcFOS increases faecal sIgA

level during the first 6 months of life

P. Scholtens et al., J Nutr 2008; 138: 1141-1147

sIgA increment:

-better first line defence

-less infections

-less allergies

-tolerance induction

Placebo scGOS/lcFOS Breastfed0

250

500

750

22

24

31

Faecal

sIg

A

(g

/g)

P 0.001

Bruzzese study

Open, placebo-controlled, randomised, prospective study 324 healthy infants

gestational age between 37 and 42 weeks (birth weight appropriate for

gestational age)

Feeding regime formula feeding for 12 months

Formulas Standard formula (n =173)

Standard formula plus 0.4 g/100 ml scGOS/lcFOS (n=169)

Outcome parameters Primary outcome: incidence of acute diarrhoea, upper- and lower respiratory

tract infection, use of antibiotics, number of hospital admissions

Secondary outcome: nutritional adequacy

scGOS/lcFOS reduce incidence of acute

diarrhea and antibiotic courses in infants

Bruzzese et al., 2009, Clinical Nutrition, in press

Reduction of mean number of

episodes of gastroenteritis

Trend for reduced recurrence

of upper respiratory tract

infections (URTI)

Reduction of antibiotics

courses used

at least 1 URTI 3 URTI0

10

20

30

40

50

6065 Placebo

scGOS/lcFOS

65/10960/94

29/65

17/60

% o

f ch

ild

ren

wit

h U

RT

I

0

25

50

75

100 Placebo

scGOS/lcFOS

At least 1 antibiotic

course 2 antibiotic

course

% o

f ch

ild

ren

wit

h a

nti

bio

tics

58/65

46/60

43/65

24/60

P=0.06 p<0.02

Placebo scGOS/lcFOS0.0

0.1

0.2

0.3

0.4

0.5

Nu

mb

er

of

ep

iso

des/c

hil

d/1

2 m

on

ths

p<0.01

Pathogens

Viruses

Parasites

Fungi

Proteins

mechanical defencee.g. skin, mucosa, mucus layer

chemical defencee.g. SCFA, acidic pH

biological defencehealthy microbiota prevents growth

of pathogens

immunological defencee.g. antibodies, cytokines,

white blood cells,

macrophages

Conclusion

Pathogens

Viruses

Parasites

Fungi

Proteins

mechanical defencee.g. skin, mucosa, mucus layer

chemical defencee.g. SCFA, acidic pH

biological defencehealthy microbiota prevents growth

of pathogens

immunological defencee.g. antibodies, cytokines,

white blood cells,

macrophages

Unique mixture of

Prebiotics

Conclusion

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