INTERACTION OF AN ASTHMA PROMOTING IL4RA ALLELE WITH OXIDATIVE STRESS PATHWAYS

Loida Viera-Hutchins, M.D.Mentor: Talal Chatila, M.D., MSc.

Overall objective

To study the role gene-environment interactions in promoting the development of asthma

Asthma

• 20 million• 10 million allergic asthma • Increase in the prevalence 75% from 1980-

1994• Children < 5 asthma rates increased >160%

from 1980-1994

EnvironmentalFactors

Geneticpolymorphism

Inter-individual variability

Asthma

Air Pollution

Ozone (O 3) Particles Sulfur Dioxide (SO ) sulfur Oxides of Nitrogen (NO x) Volatile Organic Compounds (VOCs)

The impact of particulate pollutants on asthma

Cardiorespiratory morbidity and mortality Asthma flares1. Increased symptom score 2. Requirement for more frequent medication3. Hospitalization

Particulate pollutants & allergic sensitization

Children who live near motorways have increased incidence asthma

In humans intranasal co-administration of Diesel exhaust particles (DEP) and neo-antigen (KHL) → primary sensitization and anti-KHL specific IgE in 9 of 15 atopic patients

J Allergy Clin Immunol 1999;1183-8

Murine ModelsParticle exposure during antigen sensitization increases:

Airway hyper-reactivity Airway inflammatory cells Number of goblet cells Antigen specific IgE levels Increase in pro-allergic T helper 2 cytokine

profile (Th2) IL-5, IL4, IL-13 Decrease in T helper 1 cytokine IFN-g

Particles

Coarse 2.5–10 µm Fine ≤2.5 µm Ultrafine ≤0.1 µm Diesel Exhaust Particles (DEP)

(composed of fine and ultrafine particles)

Particle Composition

Free Radic Biol Med. 2008 May 1; 44(9): 1689–1699.

*Organic Carbons: polycyclic aromatic hydrocarbons (PAH) and quinones

*

Role of oxidative stress in the health effects of particulate pollutants

Oxidative stress is a state of redox disequilibrium

Decrease in the cellular glutathione (GSH)/glutathione disulfide (GSSG) ratio

Activates a number of the redox-sensitive signaling cascades

Responses that could be protective or injurious in nature

Oxidative stress promotes dendritic cell pro-allergic Th2 skewing

DEP induced oxidative stress inhibits TH1 immunity in response to TLR agonist

TH1 immunity restored with administration of antioxidant, N-acetylcysteine

Clin Immunol. 1999 Dec;104(6):1183-8.

EnvironmentalFactors

Geneticpolymorphism

Inter-individual variability

Asthma

IL4Rαpolymorphism, Q576R

Severe asthma Severe RSV bronchiolitis Rapid decline in lung function in smokers Heightened allergen sensitization in the context

of maternal smoking 70% allele frequency in African Americans vs.

20% in Caucasians, 50% and 4% homozygosity, respectively

Q576R mutation promotes intense allergen-induced airway inflammation and remodeling

Increased peribronchial and perivascular inflammation

Increased goblet cell Increased bronchoalveolar lavage (BAL)

fluid eosinophils Sub-epithelial cell fibrosis Augments IL-4R –dependent signaling

J Exp Med. 2009 Sep 28;206(10):2191-204.

Specific Aim Study the impact of Q576R X Diesel

exhaust particles (DEP) interaction on allergen induced airway disease.

Hypothesis DEP acts as an adjuvant to promote

allergic airway sensitization Q576R synergizes with DEP exposure to

promote heightened allergic airway inflammation

In-vivo study design:6-8 week old

WT Q576RIntranasal Sensitization1.Saline2.UFP3.OVA4.UFP+OVA

3 day 1% OVA aerosol challenge

Study DesignWT Q576R

Total IgE & OVA-IgE ELISA

Bronchoalveolar lavage (BAL):Total cell # & diffIL-4, IL-13,IL-6 IL-17A, INF-γ

Lung histochemical analysis, PAS staining

In-vitro studies

DEP acts as an adjuvant to promote allergic airway sensitization

Mechanism of DEP associated allergic sensitization

Oxidative stress Prelim data: Chatila lab performed gene

microarray of DEP exposed human dendritic cells

Increase in genes in the oxidative stress pathway

Increase in Jagged1

Notch Th1 vs. Th2

Nat Rev Immunol. 2009 Feb;9(2):116-24.

Notch pathway and asthma

May program cells toward proallergic Th2 vs Th1 pathways

Jagged 1 or Jagged 2 + Notch 1 or 2 Th 2 DLL1 or DLL4 + Notch3 Th 1 Administration of Notch pathway inhibitor, Gamma

Secretase Inhibitor (GSI) inhibits asthma features

. Am J Respir Crit Care Med. 2009 May 15;179(10):875-82

In-vitro protocol

Murine bone-marrow

DEP X 24 hr: 2.5ug cm2 - 15ug cm2

Flow-cytometryDC confirmation& protein expression

Quantitative PCR gene expression

DC culture led to 60-70% DC purity

Dendritic cell Gate Unstained sample

CD11c+

68%

0 200 400 600 800 1000FSC-H

Data.009

R5

100 101 102 103 104FL1-H

Data.009

100 101 102 103 104FL1-H

Data.010

DC culture treatment with DEP results in up to 20X increase in Jagged 1 expression

0 2.5 5 10 15 20units ug/cm2, n=3 per group

DC culture treatment with DEP results in 40% decrease in Notch 1 expression

0 2.5 5 10 15 20units ug/cm2, n=3 per group

DC culture treatment with DEP results in a reduction of DLL1

0 2.5 5 10 units ug/cm2, n=3 per group

DLL1

0 5 ug/cm20.00

0.25

0.50

0.75

1.00

Treatment Group(n=3, Mann-Whitney test, p = 0.1)

No difference in Jagged 2 or Notch 2 gene expression

Jagged 2 Notch 2

No difference in Notch 3 or Notch 4 gene expression

Notch 3 Notch 4

No difference in DLL4 or DLL3 gene expression

DLL4 DLL3

0 200 400 600 800 1000

Data.001

R3

Unstained Control

100 101 102 103 104FL1-H

Data.003

R5

CD11c+ Gate

Flow results

FSC

ssc

DEP treatment results in suppression of DLL1

Gate CD11c+Red: DEP treatedBlue: UntreatedPurple: Negative Control

n=3100 101 102 103 104

DLL4 APC

DEP treated group decreased Notch 2

Gate CD11c+

Red: DEP treatedBlue: UntreatedPurple: Negative Control

n=3100 101 102 103 104

DLL4 APC

No difference in Notch 3 and DLL4

100 101 102 103 104FL4-H

Notch 3 DLL4

100 101 102 103 104FL4-H

Ongoing experiments

PCR: IL-2, IL-4, IL-13,IFN-gamma, GATA-3,

T-bet

DEPtxd +

DO11 T cells

Tx OVA

Proliferation, CFSE

Summary

Preliminary results suggest that a potential mechanism by which DEP promotes allergic sensitization TH2 DC programming via the Notch pathway

Assess for differences in the Q576R mice Confirm these results in-vivo

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