INTERACTION OF AN ASTHMA PROMOTING IL4RA ALLELE WITH OXIDATIVE STRESS PATHWAYS Loida Viera-Hutchins, M.D. Mentor: Talal Chatila, M.D., MSc.
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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References1. Rangasamy, T., et al. 2005. Disruption of Nrf2 enhances susceptibility to severe
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2. Raffi Tachdjian1, Clinton Mathias3, Shadi Al Khatib1, Paul J. Bryce3, Hong S. Kim1, Frank Blaeser4, Brian D. O'Connor2, Danuta Rzymkiewicz1, Andrew Chen1, Michael J. Holtzman5, Gurjit K. Hershey6, Holger Garn7, Hani Harb7, Harald Renz7, Hans C. Oettgen3, and Talal A. Chatila1 Pathogenicity of a disease-associated human IL-4 receptor allele in experimental asthma. J Exp Med. 2009 Sep 28;206(10):2191-204.
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5. Am J Respir Crit Care Med. 2009 May 15;179(10):875-82.