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  • 12/22/13

    1

    The Surprising Role of IFN- in AERD

    Larry Borish, M.D. Professor of Medicine Asthma and Allergic Disease Center University of Virginia Charlottesville, VA

    Conflict of Interest

    Grant Support: NIH, Dupont

    Consulting Work: Endo, Allakos, PluriStem,

    Genentech, Novartis

    Honoraria: Merck

    Acknowledgements

    University of Virginia

    John Steinke, PhD

    Lixia Liu, MD

    Julie Negri, BS

    Thomas Braciale, MD/PhD

    Spencer Payne, MD

    Phil Huyett, MD

    Josh Kennedy, MD

    Brigham and Womens Hospital

    Tanya Laidlaw, MD

    Joshua Boyce, MD

    Northwestern University

    Bruce Bochner, MD

    University of Illinois

    Steven Ackerman, PhD

    Learning Objectives

    to understand inflammatory pathways of AERD, in particular roles of novel leukotriene signaling pathways

    to understand mechanisms of aspirin/NSAID triggering of non-IgE mediated anaphylaxis in AERD

    AERD as a Disease of Excessive Cysteinyl Leukotriene Production

    and Responsiveness Constitutive over-production of CysLTs

    Over-expression of CysLT receptors

    Surge in CysLT production on exposure to

    aspirin and other non-selective (cox-1) inhibitors

    Protection from the anaphylactoid response via

    LT modifiers

    Therapeutic Implications

    LTE4 Increases After Aspirin Challenge in Aspirin Sensitive Subjects

    Urinary LTE4 following aspirin (closed symbols) and placebo (open symbols) in aspirin-intolerant (circles) and aspirin tolerant (triangles) subjects

    Christie, P.E., et al. Am. Rev. Respir. Dis. 1991;143:1025-9

  • 12/22/13

    2

    Responsiveness of subjects with AERD to LTE4 pre and post desensitization

    Arm JP et al. Am Rev Respir Dis 1989; 140:148-53

    Hyperresponsiveness was not observed for LTC4

    Christie PE Eur Respir J 1993;6:1468-73

    AERD Therapeutic Implications

    Aspirin Desensitization

    Leukotriene Modifiers

    Leukotriene receptor antagonists (e.g., montelukast, zafirlukast) improve lung function, decrease rescue bronchodilator use, reduce symptoms, and improve qol

    Zileuton may be uniquely effective including in reducing upper airway symptoms

    Dahln B et al., Am J Resp Crit Care Med 157: 1187, 1997

    Aspirin Desensitization Treatment for AERD: Patients treated 1-3 yrs (n=29)

    Baseline

    ASA Rx

    Median

    Range

    Median

    Range

    p value

    Sinusitis

    6

    1-12

    2

    0-12

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    3

    I: Too many eosinophils:

    200x 100x

    Payne, S., et al. Laryngoscope 2011

    Eosinophils

    0

    1

    2

    3

    4

    Control (7)

    Inflammatory (19)

    AFS (6)

    CHES (21)

    AERD (8)

    Eosinophil Score

    0.14

    0.58

    1.93

    1.86

    3.54

    Control (7)

    Inflammatory

    (19)

    AFS (6)

    CHES (21)

    AERD (8)

    p=n.s. p

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    4

    Why the Upregulation of Cysteinyl Leukotrienes in AERD:

    Role of Cytokines

    Th1/Th2 Cytokine Signature in Chronic Sinusitis (qPCR)

    (n=9)

    (n=30)

    (n=15)

    2CT

    x1

    0-2 (

    IL-4

    , IL-

    13);

    x10-

    4 (IF

    N-

    ); x1

    0-5 (

    IL-5

    )

    0

    20

    10

    40

    30

    50

    IL-4

    IL-5

    IL-13

    IFN-

    Steinke J., et al. 2013. J. Allergy Clin Immunol., in press

    Image Stream

    Side Scatter

    DAPI

    CCR3 PE

    Bright

    Field

    DAPI/CCR3

    Steinke J., et al. 2013. J. Allergy Clin Immunol., in press

    Intracellular Cytokine Staining of CCR3 +ve Eosinophils in CHES

    IL-5

    APC

    IL-4

    APC

    IFN

    - A

    PC

    CCR3 - PE

    Steinke J., et al. 2013. J. Allergy Clin Immunol., in press

    Intracellular Cytokine Staining of CCR3 +ve Eosinophils in AERD

    CCR3 - PE

    IL-5

    APC

    IL-4

    APC

    IFN

    - A

    PC

    Steinke J., et al. 2013. J. Allergy Clin Immunol., in press

    Eosinophil Intracellular Cytokine Expression

    CHES AERD IL-4 33.28.8 34.810.1 IFN- 15.03.4 28.210.7

    Steinke J., et al. 2013. J. Allergy Clin Immunol., in press

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    5

    i

    i

    isotype IFN-

    IFN- Immunofluorescence: CHES

    Steinke J., et al. 2013. J. Allergy Clin Immunol., in press

    IFN- Immunofluorescence: AERD

    i

    i

    isotype IFN-

    Steinke J., et al. 2013. J. Allergy Clin Immunol., in press

    IL-4 Regulates IgE-dependent CysLT production by Mast Cells: Profound Induction of LTC4 Synthase expression by IL-4:

    Hsieh FH et al. J. Exp Med. 2001;193:123.

    Effect of IL-4 priming of human MCs on IgE mediated CysLT release

    Effect of IL-4 priming on 5-LO/LTC4S pathway protein expression by hMCs

    Cytokines NOT Associated With Upregula=on of LTC4S mRNA in Eosinophils:

    IL-4

    IL-5

    IL-13

    GM-CSF

    IFN- Poten=a=on of Eosinophilopoiesis

    IL-5/IL-3 x 3 weeks 400x

    + IFN-

    CD34+ hematopoietic stem cells cultured for 3 days with SCF, TPO, Flt3L, IL-3, and IL-5 then 3 weeks with only IL-3/IL-5 IFN-

    Steinke J., et al. 2013. J. Allergy Clin Immunol., in press

    Eosinophil cellSamples_E-IFNy.fcsEvent Count: 7870

    0 102 103 104 105: Siglec 8

    0

    102

    103

    104

    105

    : C

    CR

    3

    5.81 4.19

    4.3185.7

    Eosinophil cellSamples_E+IFNy.fcsEvent Count: 7907

    0 102 103 104 105: Siglec8

    0

    102

    103

    104

    105

    : C

    CR

    3

    1.83 8.56

    3.6585.9

    Eosinophil cellSamples_M-IFNy.fcsEvent Count: 6143

    0 102 103 104 105

    0

    102

    103

    104

    105

    1.79 7.5

    11.379.4

    Eosinophil cellSamples_M+IFNy.fcsEvent Count: 4088

    0 102 103 104 105

    0

    102

    103

    104

    105

    1.44 19

    9.1570.5

    -IFN- +IFN-

    Siglec 8 Siglec 8

    CCR3

    CCR3

    Enhanced eosinophilopoiesis in additional presence of IFN-:

    Steinke J., et al. 2013. J. Allergy Clin Immunol., in press

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    6

    Enhanced Eosinophilopoiesis by IFN-

    0

    5

    10

    15

    20

    25

    30

    35

    Sigl

    ec8+

    /CCR

    3+ (%

    )

    -IFN-

    +IFN-

    Steinke J., et al. 2013. J. Allergy Clin Immunol., in press

    IFN- Activation of Eosinophilopoiesis

    Siglec8 ECP CysLT1 CysLT2 P2Y12 EP2 LTC4S Cox2 PgD2S -IFN-

    .0063

    .0017 .109 .030

    .052

    .024 .00059 .00031

    .0068

    .0021 .072 .032

    .033

    .014 .0043 .0013

    .059

    .023 +IFN- .018

    .006 .093 .018

    .076

    .027 .00053 .00012

    .0144

    .0042 .047 .015

    .080

    .021 .0183 .0134

    .220

    .072 fold increase

    2.88* .85 1.46* .90 2.11* .66 2.38* 4.23* 3.75*

    qPCR data reflect relative expression in comparison to house-keeping gene (EF1) (2CT) (n=12)

    Fold increase induced by IFN- in comparison to IL-5 and IL-3 alone

    *p

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    7

    IL-4 Induction of CysLT1 and 2 Receptor Expression

    Early, S.B. et al. Am J. Respir Cell Mol. Biol. 2007;36:715-20

    IL-4-induced CysLT1 and 2 receptor expression on T and B cells

    Cytokine Induction of CysLT 1 Receptor Protein on T cells

    Uns

    timul

    ated

    + IL

    -4

    + IF

    N-

    81 kd

    38.5 kd

    Early, S.B., et al. Am. J. Resp. Cell Mo. Biol. 36(6):715-20

    Why dont aspirin tolerant or even non-asthmatics respond to a reduction in PgE2

    (what is the mechanism of cellular activation)?

    Aspirin-Induced Eosinophil Ca+2 Flux

    0

    1000

    2000

    3000

    4000

    5000

    6000

    7000

    0 .3 1 3 10 0 .3 1 3 10 0 .3 1 3 10

    Aspirin TolerantAERD

    [PgD

    2] n

    g/m

    l

    0

    1000

    2000

    3000

    4000

    5000

    6000

    7000

    0 .3 1 3 10 0 .3 1 3 10 0 .3 1 3 10

    Aspirin TolerantAERD

    [PgD

    2] n

    g/m

    l

    0

    1000

    2000

    3000

    4000

    5000

    6000

    7000

    0 .3 1 3 10 0 .3 1 3 10 0 .3 1 3 10

    Aspirin TolerantAERD

    [PgD

    2] n

    g/m

    l

    Calc

    ium

    Flu

    x

    (a

    rbitr

    ary

    units

    )

    LysASA

    NaSalicylate

    p

  • 12/22/13

    8

    EDN (ng/10

    5 eosinophils)

    -IFN-

    +IFN-

    Medium Medium LysASA LysASA PMA/ion PMA/ion

    1 mM 1 mM 10 mM 10 mM

    *

    *

    *

    *

    *p