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INTERACTION OF AN ASTHMA PROMOTING IL4RA ALLELE WITH OXIDATIVE STRESS PATHWAYS Loida Viera-Hutchins, M.D. Mentor: Talal Chatila, M.D., MSc.

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Presentation on theme: "INTERACTION OF AN ASTHMA PROMOTING IL4RA ALLELE WITH OXIDATIVE STRESS PATHWAYS Loida Viera-Hutchins, M.D. Mentor: Talal Chatila, M.D., MSc."— Presentation transcript:

1 INTERACTION OF AN ASTHMA PROMOTING IL4RA ALLELE WITH OXIDATIVE STRESS PATHWAYS Loida Viera-Hutchins, M.D. Mentor: Talal Chatila, M.D., MSc.

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

3 Asthma 20 million 10 million allergic asthma Increase in the prevalence 75% from Children 160% from

4 Environmental Factors Environmental Factors Genetic polymorphism Genetic polymorphism Inter-individual variability Asthma

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

6 The impact of particulate pollutants on asthma Cardiorespiratory morbidity and mortality Asthma flares 1. Increased symptom score 2. Requirement for more frequent medication 3. Hospitalization

7 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

8 Murine Models Particle 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

9 Particles Coarse 2.5–10 µm Fine ≤2.5 µm Ultrafine ≤0.1 µm Diesel Exhaust Particles (DEP) (composed of fine and ultrafine particles)

10 Particle Composition Free Radic Biol Med May 1; 44(9): 1689–1699. *Organic Carbons: polycyclic aromatic hydrocarbons (PAH) and quinones *

11 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

12 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 Dec;104(6):

13 Environmental Factors Environmental Factors Genetic polymorphism Genetic polymorphism Inter-individual variability Asthma

14 IL4R α polymorphism, 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

15 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 Sep 28;206(10):

16 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

17 In-vivo study design: 6-8 week old WT Q576R Intranasal Sensitization 1.Saline 2.UFP 3.OVA 4.UFP+OVA 3 day 1% OVA aerosol challenge

18 Study Design WTQ576R Total IgE & OVA-IgE ELISA Bronchoalveolar lavage (BAL): Total cell # & diff IL-4, IL-13,IL-6 IL-17A, INF-γ Lung histochemical analysis, PAS staining

19 In-vitro studies DEP acts as an adjuvant to promote allergic airway sensitization

20 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

21 Notch Th1 vs. Th2 Nat Rev Immunol Feb;9(2):

22 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 May 15;179(10):875-82

23 In-vitro protocol Murine bone- marrow DEP X 24 hr: 2.5ug cm ug cm 2 Flow-cytometry DC confirmation & protein expression Quantitative PCR gene expression

24 DC culture led to 60-70% DC purity Dendritic cell GateUnstained sample CD11c+ 68%

25 DC culture treatment with DEP results in up to 20X increase in Jagged 1 expression units ug/cm 2, n=3 per group

26 DC culture treatment with DEP results in 40% decrease in Notch 1 expression units ug/cm 2, n=3 per group

27 DC culture treatment with DEP results in a reduction of DLL units ug/cm 2, n=3 per group

28 No difference in Jagged 2 or Notch 2 gene expression Jagged 2Notch 2

29 No difference in Notch 3 or Notch 4 gene expression Notch 3Notch 4

30 No difference in DLL4 or DLL3 gene expression DLL4DLL3

31 Unstained ControlCD11c+ Gate Flow results FSC ssc

32 DEP treatment results in suppression of DLL1 Gate CD11c+ Red: DEP treated Blue: Untreated Purple: Negative Control n=3

33 DEP treated group decreased Notch 2 Gate CD11c+ Red: DEP treated Blue: Untreated Purple: Negative Control n=3

34 No difference in Notch 3 and DLL4 Notch 3DLL4

35 Ongoing experiments PCR: IL-2, IL-4, IL-13, IFN-gamma, GATA-3, T-bet DEP txd + DO11 T cells Tx OVA Proliferation, CFSE

36 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

37 References 1. Vercelli, D Discovering susceptibility genes for asthma and allergy. Nat Rev Immunol 8: Chatila, T.A Interleukin-4 receptor signlaing pathways in asthma pathogenesis. Trends Mol. Med. 10: Hershey, G.K.K., et al The association of atopy with a gain-of- function mutation in the a subunit of the interleukin 4 receptor. N. Engl. J. Med. 337: Ober, C., et al Variation in the Interleukin 4-Receptor alpha Gene Confers Susceptibility to Asthma and Atopy in Ethnically Diverse Populations. Am J Hum Genet 66: Howard, T.D., et al Gene-gene interaction in asthma: IL4RA and IL13 in a Dutch population with asthma. Am J Hum Genet 70: Sandford, A.J., et al Polymorphisms in the IL4, IL4RA, and FCERIB genes and asthma severity. J Allergy Clin Immunol 106:

38 References 1. Rosa-Rosa, L., et al The R576 IL-4 receptor alpha allele correlates with asthma severity. J Allergy Clin Immunol 104: Wenzel, S.E., et al IL4R alpha mutations are associated with asthma exacerbations and mast cell/IgE expression. Am J Respir Crit Care Med 175: Blaeser, F., et al Targeted Inactivation of the IL-4 Receptor {alpha} Chain I4R Motif Promotes Allergic Airway Inflammation. J Exp Med 198: Saxon, A., et al Air pollution and allergy: you are what you breathe. Nat Immunol 6: McCreanor, J., et al Respiratory effects of exposure to diesel traffic in persons with asthma. N Engl J Med 357: Whitekus, M.J., et al Thiol antioxidants inhibit the adjuvant effects of aerosolized diesel exhaust particles in a murine model for ovalbumin sensitization. J Immunol 168:

39 References 1. Rangasamy, T., et al Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice. J Exp Med 202: Raffi Tachdjian 1, Clinton Mathias 3, Shadi Al Khatib 1, Paul J. Bryce 3, Hong S. Kim 1, Frank Blaeser 4, Brian D. O'Connor 2, Danuta Rzymkiewicz 1, Andrew Chen 1, Michael J. Holtzman 5, Gurjit K. Hershey 6, Holger Garn 7, Hani Harb 7, Harald Renz 7, Hans C. Oettgen 3, and Talal A. Chatila 1 Pathogenicity of a disease- associated human IL-4 receptor allele in experimental asthma. J Exp Med Sep 28;206(10): Li N, Sioutas C, Cho A, Schmitz D, Misra C, Sempf J, Wang M, Oberley T, Froines J, Nel A. Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage. Environ Health Perspect 2003;111:455– Carine Delayre-Orthez a, Julien Becker a, Frédéric de Blay a, b, Nelly Frossard a, Françoise Pons a. Exposure to Endotoxins during Sensitization Prevents Further Endotoxin-Induced Exacerbation of Airway Inflammation in a Mouse Model of Allergic Asthma. Int Arch Allergy Immunol 2005;138:

40 References 1. Proteomics Dec Health Perspectives 2003; 111: Toxicology Letter 2004; 149: Clin Immunol Dec;104(6): Am J Respir Crit Care Med May 15;179(10):


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