CAROTENOIDS, INFLAMMATION AND ASTHMA

Dr Lisa WoodDr Lisa Wood

Centre for Asthma and Respiratory Diseases

Hunter Medical Research Institute

University of Newcastle

CAROTENOIDS –IMPORTANT DIETARY

ANTIOXIDANTSANTIOXIDANTS

ANTIOXIDANTS

ANTIOXIDANTS protect the body from the damaging effects of free radicals, preventing OXIDATIVE STRESS. Mechanisms include: scavenging of free radicals, binding of metal ions, regeneration of other AO.

3

ENDOGENOUS

ANTIOXIDANTS

eg Glutathione

GSHPx

SOD

Catalase

DIETARY

ANTIOXIDANTS

eg Vitamin C

Vitamin E

Flavonoids

Carotenoids

CAROTENOIDS

• Natural fat-soluble pigments found mainly in plants

• Red, orange, yellow pigments

• Most prevalent as trans-isomers

• Interact with lipophilic cell components eg membranes and lipid globules

• Sensitive to heat and light

• Humans cannot synthesize

• 600 carotenoids isolated from natural sources– 40 found in Western diet

– 21 found in human plasma

α-carotene: β-carotene:

Lutein:

STRUCTURE

Lycopene: Zeaxanthin:

Carotenoids

Carotenes Xanthophylls

α-carotene

β-carotene

Lutein

Zeaxanthin

CLASSIFICATION

Lycopene Cryptoxanthin

ANTIOXIDANT PROPERTIES OF CAROTENOIDS

• Singlet oxygen quenching

• Free radical quenching

– CAR + ROO• → CAR •+ + ROO- (Electron transfer)

– CAR + ROO• → CAR • + ROOH (Hydrogen abstraction)– CAR + ROO• → CAR • + ROOH (Hydrogen abstraction)

– CAR + ROO• → ROOCAR• (Addition)

• Increased activity of antioxidant enzymes

• Lycopene > α-carotene > β-cryptoxanthin

> zeaxanthin= β -carotene > lutein

FOOD SOURCESFood ββββ-carotene (µg/100

g):αααα-carotene Lutein+Zeaxanthin Lycopene

Apricot 3524 0 0 5

Broccoli 700 1 1900 0

Carrot 7900 3600 260 0

Mango 1300 0 0 0

Nectarine 103 0 NA NA

Peach 99 1 14 0

Pumpkin 3100 3800 1500 0

Spinach 4100 0 10200 0

Tomato paste 1700 NA NA 6500

Tomato juice 900 NA NA 8500

Tomato raw 520 NA 100 3100

Watermelon 230 1 14 4100

(Mahan, 2004)

BIOAVAILABILITY

Fat essential for lycopene uptake:

Subjects had ingested a fresh vegetable salad with salad dressing containing:▼ 0g(fat-free),

○ 6g(reducedfat), or

● 28g (full-fat) canola oil; n=7.

(Brown, AJCN, 2004)

ASTHMA –A CHRONIC INFLAMMATORY DISEASE OF THE AIRWAYS

ASTHMA PREVALENCE

� Prevalence: increased over time, higher in Westernised countries � Migration from developing to Western country can lead to asthma (Gibson, 03)

ENVIRONMENTAL FACTORS INFLUENCING

DEVELOPMENT AND EXPRESSION OF ASTHMA

Allergens: Indoor (dust mites, dogs, cats, cockroaches)Outdoors (pollens, molds, fungi, yeasts)

Infections (early childhood)Occupational exposureSmoking (active/ passive)

Diet = Antioxidants, Dietary Fat, Obesity

(GINA, 2004)

ANTIOXIDANTS AND ASTHMA

� Oxidative stress occurs in asthma because..asthmatics exhibit an exaggerated immune response to stimuli (e.g. allergens and viruses). Airway inflammatory cells respond with a ‘respiratory burst’, involving oxygen cells respond with a ‘respiratory burst’, involving oxygen uptake and leakage of free radicals into surrounding cells.

INFLAMMATORY CELL ACTIVATION

ACQUIRED IMMUNITY INNATE IMMUNITY

AllergenNO2/Ozone Chemicals

Activated TH2 cells

IL-5

Eosinophils

IL-8

Neutrophils

Activated epithelial cells/macrophages

Virus Endotoxin

MECHANISMS LEADING TO OXIDATIVE STRESS IN ASTHMA

OXIDATIVE STRESS

ASTHMA

Airway Hyperresponsiveness, Smooth Muscle Contraction, Mucus Hypersecretion, Epithelial Cell Shedding

Mediator Release including ROS (O2.-, H2O2,

.OH)

Eosinophils Neutrophils

2

(Wood et al; ERJ, 2003)

N

EFFECTS OF OXIDATIVE STRESS IN ASTHMA

Air enters respiratory tract via mouth

and nose and enters bronchial tubes

ASTHMA:

Inflamed bronchial tube NORMAL

bronchial tube

Smooth muscle contracts and becomes ‘twitchy’

Excess mucus

500

1000ta

ne (

pg/m

L)

OXIDATIVE STRESS IN ASTHMA

Controls

Healthy

Asthma

Stable Asthma

Post Exac

0

8-isopro

st

Exac

Asthma

(Wood, AJRCCM, 2005)

INFLAMMATORY CELL ACTIVATION

ACQUIRED IMMUNITY INNATE IMMUNITY

AllergenNO2/Ozone Chemicals

Activated TH2 cells

IL-5

Eosinophils

IL-8

Neutrophils

Activated epithelial cells/macrophages

Virus Endotoxin

MECHANISMS LEADING TO OXIDATIVE STRESS IN ASTHMA

OXIDATIVE STRESS

ASTHMA

Airway Hyperresponsiveness, Smooth Muscle Contraction, Mucus Hypersecretion, Epithelial Cell Shedding

Mediator Release including ROS (O2.-, H2O2,

.OH)

Eosinophils Neutrophils

2

(Wood et al; ERJ, 2003)

Antioxidant supplementation

quercetincatechinsphloridzin

Cough, wheeze (Tabak, 2001)Asthma (Shaheen,2001)FEV1 (Butland, 1999)

Apples/pears

vitamin Ccarotenoidsflavonoids

Wheeze (Butland, 99)FEV1 (Cook,97; Carey,98)Chronic lung disease (Miedema,93)

Fresh fruit

Active AO?Respiratory MarkerAO-rich Food

quercetincatechinsphloridzin

Cough, wheeze (Tabak, 2001)Asthma (Shaheen,2001)FEV1 (Butland, 1999)

Apples/pears

vitamin Ccarotenoidsflavonoids

Wheeze (Butland, 99)FEV1 (Cook,97; Carey,98)Chronic lung disease (Miedema,93)

Fresh fruit

Active AO?Respiratory MarkerAO-rich Food

EPIDEMIOLOGY OF ANTIOXIDANT-RICH FOODS AND ASTHMA

carotenoidsflavonoids

Wheeze (Ellwood,01; Hijazi,00)Bronchitis/asthma (LaVecchia,97)

Vegetables and Vegetable prod

lycopenequercetin

Asthma onset (Troisi, 1995)Tomato prod (pste/sce/pizza)

Vit E, SeWheeze (Ellwood, 01)Cereals

anthocyaninquercetin

Asthma severity (Shaheen, 01)Red Wine

carotenoidsflavonoids

Wheeze (Ellwood,01; Hijazi,00)Bronchitis/asthma (LaVecchia,97)

Vegetables and Vegetable prod

lycopenequercetin

Asthma onset (Troisi, 1995)Tomato prod (pste/sce/pizza)

Vit E, SeWheeze (Ellwood, 01)Cereals

anthocyaninquercetin

Asthma severity (Shaheen, 01)Red Wine

It is probable that a combination of nutrients are

providing protective effect

PLASMA CAROTENOID LEVELS IN ASTHMA

400500600700800900

100011001200

200

300

400

500(υg/L) (µg/L)

** * *

Asthma

Controls

0100200300400

0

100

**

* **

*

Total Carotenoids

lutein β-crypto-xanthin

lycopene α-carotene β-carotene

Plasma carotenoids reduced in asthma despite no differences in dietary intake

(Wood et al; JACN, 2005)

INVESTIGATINGLYCOPENE SUPPLEMENTATION TO PROTECT AGAINST ASTHMA

TRIGGERSTRIGGERS

VIRUSES ALLERGY

TRIGGER: Allergy

day -14 0 12 13 14 15 16

ip OVA sacrificeCommence daily nutrient

supplementation in OVA

Mice were sensitized by

intraperitoneal (ip) injection of 50 µg of OVA

Mice were challenged by intranasal (in) instillation of 10 µg of OVA

naive PBS OVA-con OVA-lyc0

1

2

3

4

5

6

7

8

9

*

* *

eo

sin

op

hil

s/m

L B

AL

F (

x10

4)

TRIGGER: Allergy

naive PBS OVA-con OVA-lyc0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

*

* *

blo

od

eo

sin

op

hil

s (

%)

40

naive PBS OVA-con OVA-lyc0

5

10

15

20

*

* *eo

sin

op

hil

s/1

00

µµ µµM

air

way

naive PBS OVA-con OVA-lyc0

10

20

30

40

*

* *

MS

C/1

00

µµ µµM

air

way

(Hazelwood et al, submitted July 2009)

Supplementation reduced eosinophilic infiltrates in the bronchoalveolar lavage fluid, lung tissue and blood, and mucus secreting cell numbers in the airways.

0.1

0.2

0.3

*

* *

IL-4

(n

g/m

L)

20

30

40

*IL-5

(n

g/m

L)

TRIGGER: Allergynaive

PBS

OVA-con

OVA-lyc

unstimulated OVA-stimulated0.0

0.1 *

IL-4

(n

g/m

L)

unstimulated OVA-stimulated0

10*

* *

IL-5

(n

g/m

L)

(Hazelwood et al, submitted July 2009)

Supplementation reduced ovalbumin-specific release of Th2-associated cytokines IL-4 and IL-5 was also reduced.

100

150

200

250 P = 0.026P = 0.002

IL-6

concentr

ation (pg/m

l)

1 0 0

1 5 0

2 0 0

2 5 0 p = 0 .0 0 2 p = 0 .0 0 1

(a)

IP-1

0 c

oncentration (pg/m

l)I

TRIGGER: Virus Infection

Cel

l alo

ne

Cel

l + R

V-1B

Cel

l+0.

5% T

HF +

RV-1

B

Cel

l+0.

5% T

HF/L

ycopen

e + R

V-1B

0

50

IL-6

concentr

ation (pg/m

l)

Cel

l alo

ne

Cel

l + R

V43

Cel

l+0.

5% T

HF +

RV43

Cel

l+0.

5%TH

F/Lyc

opene+

RV43

0

5 0

IP-1

0 c

oncentration (pg/m

l)I

(b )(Saedisomeolia, J Nutr Biochem, 2009)

Pre-incubation of airway epithelial cells with lycopene (dissolved in THF):- 24% reduction in IL-6 after rhinovirus-1B infection- 31% reduction in IP-10 after rhinovirus-43 infection

500000

1000000

1500000

2000000

2500000

p =0.18

p = 0.025

TC

ID50 /

ml

TRIGGER: Virus Infection

Cel

l alo

ne

Cel

l +TH

F/lyco

pene

Cel

ls +

RV43

Cel

ls +

RV43

+ T

HF/ly

copen

eC

ells

+ R

V1B

Cel

ls +

RV1B

+ T

HF/ly

copen

e

0

(Saedisomeolia, J Nutr Biochem, 2009)

Pre-incubation of airway epithelial cells with lycopene (dissolved in THF):- 85% reduction in rhinovirus-1B replication.

INVESTIGATINGMANIPULATION OF DIETARY

ANTIOXIDANT INTAKE IN ANTIOXIDANT INTAKE IN ASTHMA

STUDY DESIGN: Subjects advised to:

1. Limit Vegetable/salad intake - 2 serves per day 2. Limit Fruit (fresh, dried, tinned) intake - 1 serve/day

3. Replace whole grain breads with processed products4. Avoid antioxidant additives (300 – 322)

5. Avoid high antioxidant foods eg. nuts, tea, red wine

DIETARY ANTIOXIDANT DEPLETION

1. Carotenoid levels reduced

00.20.40.6

0.81

1.2

*

*** *

*p<0.05

0

Total

Car

oten

oids

Lute

in/ Z

eaxa

nthi

n

beta

-cry

ptox

anth

in

Lyco

pene

alph

a-ca

rote

nebe

ta-c

arote

ne

Baseline Follow ing 10 days on Low Antioxidant Diet

*

2. Airway inflammation worsened

90

80

70

60

50

%S

pu

tum

Ne

utr

op

hils

P=0.038

Low AO Diet

50

40

30

20

10

0

Baseline

%S

pu

tum

Ne

utr

op

hils

(Wood, Free Rad Res, 2008)

95

100

105

110

Baseline Low AO Diet

1

1.2

1.4

1.6

1.8

3. Asthma worsened%

pre

dic

ted

Lu

ng

Fu

nctio

n

Asth

ma

Co

ntr

ol S

co

re

P=0.004

P=0.002

P=0.035

60

65

70

75

80

85

90

95

0

0.2

0.4

0.6

0.8

1

% p

red

icte

d L

un

g F

un

ctio

n

%FEV1 %FVC

Asth

ma

Co

ntr

ol S

co

re

(Wood, Free Rad Res, 2008)

GENOMICS OF ANTIOXIDANT WITHDRAWAL IN ASTHMA

Gene profiling of sputum samples following dietary antioxidant withdrawal

104 genes differentially expressed 22 up, 82 down.

Upregulated genes:Innate Immune Receptors (TLR2, IL1R2, CD93, ANTXR2)

Signalling Molecules (IRAK2, IRAK3, MAP3K8)

Proteases (MMP25, CPD)

Regulation of Apoptosis (CFLAR)

Visit 1 (red) V2 (blue)

(Baines, OMICS, 2009)

• Downregulated genes:– Glutathione metabolism (GSTA1, GSTA2)

– Protease Inhibitors (SLPI, SERPINB3)

– Eosinophil and Mast Cell Responses (CLC, TPSAB1)

• 5 genes including IRAK2, IL1R2, TLR2, SERPINB3, C20orf114 confirmed to be differentially expressed by real time PCRreal time PCR

-8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4

Microarray

qPCR

Fold Change from Baseline

TLR2

IL1R2

IRAK2

C20orf114

SERPINB3

(Baines, OMICS, 2009)

Plasma Antioxidants vs airway IRAK3 Gene Expression

IRAK3 gene expression is:

- Correlated with Total plasma antioxidants

- Increased by antioxidant withdrawal

- Increased in sputum in

13

14

15

To

tal A

ntio

xid

an

ts

- Increased in sputum in neutrophilic asthma

- Reported to be involved in tolerance to LPS and immunosuppression in sepsis

- Reported to be associated with early onset persistent asthma

11

12

To

tal A

ntio

xid

an

ts

-1 -.5 0 .5 1IRAK3

r=0.83

p=0.0029

(Baines, OMICS, 2009)

Wash

out

Treatment 1

Wash out

Treatment 2

Washout

Treatment 3

STUDY DESIGN:

LYCOPENE SUPPLEMENTATION IN STABLE ASTHMA

Visit 1 2 3 4 5 6 7

VS VS PLACEBO

Placebo Juice Extract0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

p<0.01

p<0.001

Ly

co

pe

ne

( u

g/m

L)

Placebo Juice Extract0

25

50

75

100

p<0.05

p<0.05

Neu

tro

ph

il E

lasta

se A

cti

vit

y (

ng

/mL

)%

Neu

tro

ph

ils

� Increased plasma lycopene

� Reduced airway neutrophils

� Reduced airway NE activity

Placebo Juice Extract0

1000

2000

3000

p<0.05

45008500

12500

Neu

tro

ph

il E

lasta

se A

cti

vit

y (

ng

/mL

)

(Wood, Free Rad Res, 2008)

-20

-10

0n F

EV

1

xerc

ise

*

STUDY DESIGN: 30 mg /day lycopene for 7 days, n = 20

LYCOPENE SUPPLEMENTATION IN EXERCISE-INDUCED ASTHMA

Before

Treatment

Follow ing

Treatment

-50

-40

-30

-20

% C

hange in

follo

win

g e

x

* p<0.05(Neuman, Allergy, 2000)

CONCLUSIONS AND FUTURE DIRECTIONS

• Low intake of antioxidant-rich foods, such as fruit and vegetables, may be contributing to increased asthma prevalence.asthma prevalence.

• Antioxidant supplementation, in particular lycopene supplementation may be useful in asthma.

• Large scale trials are needed to establish the clinical efficacy of lycopene supplementation in asthma.

ACKNOWLEDGEMENTS

• Prof Peter Gibson• Prof Manohar Garg• Prof Paul Foster• A/Prof Phil Hansbro• A/Prof Peter Wark• Dr Katie Baines• Dr Katie Baines• Dr Ahmad Saedisomeolia• Leia Hazelwood• Heather Powell

• Centre for Asthma and Respiratory Diseases, Clinical and Laboratory Teams, HMRI, John Hunter Hospital and David Maddison Building, University of Newcastle