Role of Prebiotics in Respiratory Immunity

Anang Endaryanto
Division of Allergy Immunology Department of Child Health Faculty of Medicine - University of Airlangga Dr. Soetomo General Hospital

What is immunity? (1)

Doan et. al. 2008. Lippincotts Illustrated Reviews: Immunology

What is immunity? (2)

Pathogens
Viruses

mechanical defence
e.g. skin, mucosa, mucus layer

chemical defence
e.g. SCFA, acidic pH

Fungi
Parasites Proteins

biological defence
healthy flora prevents growth of pathogens

immunological defence
e.g. antibodies, cytokines, white blood cells, macrophages

What is immunity? (3) Immune response = f (a) (b) (c) (d) (e)
a= immunogen stimuli b= DC subset c= recognition receptor d= microenvironment e= cytokines

Science 2001;293: 251-6

What is immunity? (4) What are major tasks of the immune system ?
1. Defence against pathogens: bacteria, viruses, parasites, fungi 2. Assistance in anti-cancer responses 3. Removal of foreign / non-self compounds 4. Inhibition self-reactive / auto-reactive responsiveness [Impairment of auto-immunity (SLE, MS, RA,)] 5. Inhibition over-activity/allergy
Garssen J, 2009. Immune Modulating Effects of Prebiotic Carbohydrates

How does the immune system perform the major tasks ?

By immune regulation
Resistance to infections Allergy

Th1/Th17

Th0

Th2

DCs APC

Garssen J, 2009. Immune Modulating Effects of Prebiotic Carbohydrates

What is the task of immune regulation?

Immune regulation
Th1/Th2 paradigm Resistance to intracellular infections Anti-cancer Delayed type hypersensitivity reactions
Th1/Th17 Th0 Th2

Resistance to extracellular infections

Tolerance

T-cell mediated autoimmunity Graft rejection

APC

Allergy Antibody-mediated autoimmunity

Garssen J, 2009. Immune Modulating Effects of Prebiotic Carbohydrates

If immune regulation failure, what happen?

Immune regulation
Th1/Th2 paradigm

Resistance to intracellular infections

Anti-cancer Delayed type hypersensitivity reactions
Th1/Th17 Th0

Resistance to extracellular infections Tolerance

Th2

T-cell mediated autoimmunity Graft rejection

APC

Allergy Antibody-mediated autoimmunity

Garssen J, 2009. Immune Modulating Effects of Prebiotic Carbohydrates

If immune regulation failure, what happen?

Immune regulation
Th1/Th2 paradigm Resistance to intracellular infections Anti-cancer Delayed type hypersensitivity reactions
Th1/Th17 Th0 Th2

Resistance to extracellular infections Tolerance

T-cell mediated autoimmunity Graft rejection

APC

Allergy Antibody-mediated autoimmunity

Garssen J, 2009. Immune Modulating Effects of Prebiotic Carbohydrates

What is the goal of immune regulation?

Immune regulation
The extended Th1/Th2 paradigm

Th1

Th17

Th2

Treg

IFN- IL-17 IL-12 IL-22

IL-4 IL-5 IL-13

IL-10 TGF-

Garssen J, 2009. Immune Modulating Effects of Prebiotic Carbohydrates

If immune regulation failure, what happen?

Immune disorders diseases relation

HIV COPD Allergies Asthma Atopic eczema Coeliac disease Cystic Fibroses Cancer Elderly Infants

Th1 , Th2 , Th1/Th2 Th1

Th2 (type I allergy)

Th2 Th2 Th1 Th1 ? Th1 Th1 Th1 , Th2

Garssen J, 2009. Immune Modulating Effects of Prebiotic Carbohydrates

If immune regulation failure, what happen?

Immune disorders diseases relation

Hyper immune- responsiveness: Allergy Autoimmunity Chronic inflammatory diseases

Hypo immune- responsiveness: Infections Tumors/metastasis

Role in metabolic disorders/ syndrome X/ obesity Role in neurological disorders: Autism, Alzheimer, Depression, Hyperactivity Syndrome, ... Garssen J, 2009. Immune Modulating Effects of Prebiotic Carbohydrates

What is the consequence of natural imbalance of immune system?

Immune disorders : Natural imbalance

Pregnancy and neonates

Th2 directed immune system: cytokines down regulate cellular-immunity (Th1) > no rejection of the fetus and or mother tissues

CD 69+ IL 2 TNF TNF

Th 1
Failure Recurrent Miscarriages

Th 2
Normal Pregnancies

CD 94+ IL 4 IL 5 IL 10

After birth
Development of their own healthy Th1/ Th2 balanced immune system
Archives of Disease in Childhood 2004;89:97-102

How does the Th1/Th2 balanced immune system develop?

Th1 and Th2 activity as a function of age

Genes
Hygiene Drugs Diets Stress Hormones

10

40

50

60

70

80

90

years

Th 1

Th 2

Garssen J, 2009. Immune Modulating Effects of Prebiotic Carbohydrates

Respiratory Immunity

Respiratory System

Common Mucosal Immune System

Respiratory Immunity

Schematic representation of immune homeostasis in the airway mucosa

Respiratory Immunity
Cantana A, 2008. Pediatric Allergy, Asthma & Immunology 1 st ed : p728

Where is the most active area of immune regulation ?

Organs Cells
Epithelial cell
Granulocyte

Thymus

Macrophage / Monocyte Dendritic cell T Lymphocyte (Th1, Th2, Th3, Tr, ...) B Lymphocyte / Antibodies

Spleen
Lymph nodes Bone marrow Y Y

Natural Killer Cell

Neurons, nerves

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

What is the natural source of immune regulator?

Compounds with immunological properties in human milk Compounds with immunological properties in human milk
Anti-microbial compounds Immunoglobiuines: sIgA, SIgG, SIgM Lactoferrin, lactoferrin B and H Lysozyme Lactoperoxidase Nucleotide-hydrolizing Antibodies -casein and -lactalbumin Haptocorrin Mucins Lactadherin Free secretory component Oligosaccharides and prebiotics Fatty acids Maternal leukocytes and Cytokines sCD14 Complement and complement receptors -defensin-1 Toll-like receptors Bifidus factor Tolerance/priming compounds Cytokines: Il10 and TGF Anti-idiotypic antibodies Immune development compounds Macrophages Neutrophils Lymphocytes Cytokines Growth factors Hormones Milk peptides Long-chain polyunsaturated fatty acids Nucleotides Adhesion molecules Anti-inflammatory compounds Cytokines: Il-10 and TGF Il-1 receptor anatagonist TNF and Il-6 receptors sCD14 Adhesion molecules Long-chain polyunsaturated fatty acids Hormones and growth factors Osteoprotegerin Long-chain polyunsaturated fatty acids Hormones and growth factors

Oligosaccharides

Field J Nutr 2005

The potential mechanisms of immune modulation by orally applied prebiotics. THE OLD CONCEPT
Potential mechanisms of immune modulation by orally applied non-digestible carbohydrates

Prebiotics vs Probiotics
Prebiotics Probiotics

Roberfroid MB, Gibson GR, 2008. Handbook of Prebiotics

The potential mechanisms of immune modulation by orally applied prebiotics. THE NEW CONCEPT
Major role for antigen-presenting cells

oligosaccharides

The potential mechanisms of immune modulation by orally applied prebiotics. THE NEW CONCEPT
Potential mechanisms of immune modulation by orally applied non-digestible carbohydrates
Paul Vos et al.,Critical Reviews Immunology, 2007

Evidence 1: The potential mechanisms of immune modulation by orally applied prebiotics.

Supplementation with oligosaccharides stimulate growth of bifidobacteria and lactobacilli in the intestine
log 10 of CFU/g wet faeces (median, IQR) log 10 of CFU/g wet faeces (median, IQR)

12 11

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

10 9

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

10
9 8 7 6 5 4 3

8
7 6 5 4 3 2 1
Group difference according to MannWhitney U-test: * p<0.05 vs. 0.0, # vs. 0.8

2
0
g/100ml (n=33)

Group difference according to MannWhitney U-test: * p<0.05 vs. 0.0, # vs. 0.4

0
0
g/100ml (n=33)

0.4
g/100ml (n=30)

0.8
g/100ml (n=27)

0.4
g/100ml (n=30)

0.8
g/100ml (n=27)

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

Evidence 2: The potential mechanisms of immune modulation by orally applied prebiotics.

scGOS/lcFOS increases the faecal sIgA level during the first 6 months of life

sIgA g/g faeces after 26 weeks

800

p<0,0001
700 600

24
control scGOS/lcFOS

500 400 300 200

100 0

31

breastmilk

22

P. Alliet et al., J. Pediatric Gastroenterology Nutr. 2007, 44 (ESPGHAN) A. Bakker-Zierikzee et al., Pediatric Allergy Immunol 2006; 17: 134-140

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

sIgA increment: -better first line defence -less infections -less allergies -tolerance induction

Evidence 3: The potential mechanisms of immune modulation by orally applied prebiotics.

Oligosaccharides Enhance Non-specific Immune Parameters (ex vivo)

percentage activated granulocytes

P<0.01 70 60 50 40 30 20 10 0

percentage activated granulocytes

P<0.01 55 50 45 40 35 30 25 20 15 10 5 0

Control

Enhance phagocytosis capacity Enhance oxidative burst

Jeurink et al, unpublished observations

G/lcF/lvP

Control

G/lcF/lvP

Evidence 4: The potential mechanisms of immune modulation by orally applied prebiotics.

lcFOS stimulates NO2 production by murine macrophages
60 50

lcFOS scFOS

NO2- production (uM)

40 30 20 10

0
Medium 0 0.02 0.1 0.5 2.5 5 12.5

lcFOS/scFOS (mg/ml) + IFN-

Jeurink et al, unpublished observations

Evidence 5: The potential mechanisms of immune modulation by orally applied prebiotics.

TLR9 ligand supports a regulatory Th1 type effector response; potential new synbiotic concepts

10 5

10

10 5

*
IFN- (pg/mL)

*
IL-10 (pg/mL)

*
TNF- (pg/mL)

** *

10 4

IL-6 (pg/mL)

10 4

10 3

10 4

10 3

10 3 10 2 10 1 10 0
Co ntr ol TL R2 TL R4 TL R9

10 2 10 1 10 0

10 3 10 2 10 1 10 0
Co nt ro l TL R2 TL R4 TL R9

10 2 10 1 10 0

Co ntr ol TL R Co 2 TL n Rt4 ro TL l TR LR 92 TL R4 TL R9

Kivit et al: submitted april 2009

Evidence 6: The potential mechanisms of immune modulation by orally applied prebiotics.

scGOS/lcFOS stimulates TLR9 induced IFN- production by human blood cells in co-culture systems with human gut epithelial cells
**
10 4

*** *** *

IFN- (pg/mL)
10 3 10 2 10 1 10 0

FO S S /lc R 9+ sc G O

FO S

TL

on t

TL

sc G

TL

R 4+

Kivit et al: submitted april 2009

TL

sc G

S /lc

/lc

FO S

ro l

R 4

R 9

Evidence 7: The potential mechanisms of immune modulation by orally applied prebiotics.

scGOS/lcFOS affects Th1/Th2 via DC/T-cell interaction

Ratio IFN-/IL-4 6,0 (positive cells)

5,0 4,0

**

Stimulation of anti-allergic profile 3,0

2,0 1,0

Control
GOS/lcFOS

0,0

What is the new concept of potential mechanisms of immune modulation by orally applied prebiotics?

Microbes

TLRs

!
Oligos.
C-type lect. DC-sign

Immune skewing

What is the role of FOS & GOS prebiotics in the child immunity ?

FOS

GOS/lcFOS can affect systemic

immunity (Th1/Th2/Treg) There is synergy between different types of oligosaccharides Not all oligosaccharides affect the immune system

GOS

What about improved resistance to infections ? What about reduced risk for IgE mediated allergy?

Evidence 1: Defence against pathogens.

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

Study parameters: DTH Influenza specific antibody titers

Ex-vivo lymphocyte restimulation

-20 0 7 14 21 28 30

Start dietary intervention

Blood sample Primary influenza immunization

Blood sample Booster influenza immunization

DTH End

Evidence 1: Defence against pathogens.

GOS/lcFOS improves vaccination

150 Earswelling (10 -3 mm)

100

50

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

Evidence 2: Defence against pathogens.

Oligosaccharides decrease MCMV load in vivo

Infectious virus detection in C57BL/6J liver

1 20

Control
Positive plaque assay (%)
1 00 80 60 40 20 0

Oligo

Time postinfection (days)

Evidence 3: Defence against pathogens.

Open, placebo-controlled, randomised, prospective study

O Outcomes
Incidence
1. acute diarrhoea 2. upper& lower RTI 3. use of antibiotics, 4. number of hospital admissions

I Interventions P Population
Standard formula + 0.4 g/100 ml scGOS/lcFOS per 100 ml (n=169)

Improved

Healthy infants GA37 -42 weeks BW appropriate for GA

Sample 342 infants

12 months

Not improved Improved

Control : Standard formula

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

Not improved

C Comparator

Evidence 4: Defence against pathogens.

Open, placebo-controlled, randomised, prospective study

O Outcomes
Primary outcome: serious infection morbidity Sepsis Meningitis Pyelonephritis Pneumonia

I Interventions P Population
Breast milk or Standard formula + 1.5 g/kg/day sc GOS /lcFOS/ AOS (n=54)

pre-term infants GA < 32

Sample 324 preterm infants 27 days

Improved Not improved Improved

BW 1500 gram

Control : Breast milk or Standard formula

Not improved

Westerbeek et al., 2009, submitted to ESPR

C Comparator
Enteral supplementation with oligosaccharides decrease serious infectious morbidity in preterm infants

Evidence 5: Defence against pathogens.

Protective effect on infectious episodes in High-Risk infants at 6 months

50
placebo

infectious epidsodes

40 30
p=0.01

GOS/lcFOS

20 10 0 All Infections

trend, p=0.07

URTI

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

Evidence 6: Inhibition over-activity/allergy

Allergy (respiratory)

Number of broncho alveolar cells

GOS/lcFOS impairs allergic inflammation in the lungs

400 350 300 250 200 150 100 50 0 Non-sens
P<0,001 P<0,05

Nonsens

(x 10e4)

Sens

Sens

Sens+oligo's

P.Vos, G. Folkerts, B. van Esch, G. Hofman, J. Garssen International Immunopharmacology 2007; 7(12):1582-1587.

Evidence 7: Inhibition over-activity/allergy

Allergy (respiratory)

GOS/lcFOS impairs IgE levels

100000

80000

OVA-IgE (IU/ml)

P<0,05
60000

IgG2a
40000

20000

0 No treat Oligo

P.Vos, G. Folkerts, B. van Esch, G. Hofman, J. Garssen International Immunopharmacology 2007; 7(12):1582-1587.

Evidence 8: Inhibition over-activity/allergy

Early dietary intervention with a mixture of prebiotic oligosaccharides reduce the incidence of allergic manifestations during the first 2 years of life

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

Evidence 9: Defence against pathogens & inhibition over-activity/allergy Double blind, placebo controlled, randomized prospective study O Outcomes I Interventions 1 Outcome:
st

P Population
Infants (FH+ > 1)

Non-hydrolyzed formula + 0.8g GOS/FOS/AOS per 100ml (n=416) Sample 1187 healthy infants

Incidence of Atopic dermatitis

Improved Not improved Improved

GA : 37- 42

12 mos

BW appropriate for GA

Eisses et al., 2008, Arch Dis Child. 93:A133-A135

1. Non-hydrolyzed formula (n=414) 2. Exclusive BF (n=300)

Not improved
2nd Outcome: Number of febrile episodes with a peak rectal temp 38.5C

C Comparator

Summary

Pathogens
Viruses

mechanical defence
e.g. skin, mucosa, mucus layer

chemical defence
e.g. SCFA, acidic pH

Fungi
Parasites Proteins

biological defence
healthy flora prevents growth of pathogens

immunological defence
e.g. antibodies, cytokines, white blood cells, macrophages

Non-digestible oligosaccharides (GOS/lcFOS)

Summary

Doan et. al. 2008. Lippincotts Illustrated Reviews: Immunology

GOS/lcFOS

GOS/lcFOS GOS/lcFOS

GOS/lcFOS

GOS/lcFOS

GOS/lcFOS

GOS/lcFOS

... What is the role of FOS & GOS prebiotics in the respiratory immunity ?

Immune response = f (a) (b) (c) (d) (e) a= immunogen stimuli b= DC subset c= recognition receptor FOS & GOS d= microenvironment Prebiotics e= cytokines