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Page 1: Conflict of Interest · Conflict of Interest • Grant Support: NIH, ... Effect of IL-4 priming of human MCs on ... primarily acts to block CysLT production "

12/22/13

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The Surprising Role of IFN-γ 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 Women’s 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

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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

Dahlén 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 <0.0001 Sinus surgery/yr 0.2 0-0.75 0 0-2 0.09 Hospitalizations/yr 0.2 0-0.54 0 0-1 0.005 ED visits/yr 0 0-1.22 0 0-1.5 0.2 Olfaction score 0 0-1 3 1-5 <0.0001

Asthma Response Prednisone (mg/d) 7.9 ±2.0 1.8 ±0.7 0.01 Nasal steroid (µg/day) 137 ±25.6 90 ±22.3 0.09 Inhaled steroid (µg/d) 640 ±146 885 226 0.1

Stevenson DD et al. J Allergy Clin Immunol. 1996;98:751

Decrease in Leukotriene-Receptor Expression on Nasal Mucosal Inflammatory

Cells after Aspirin Desensitization

Nasal biopsy specimens immunostained for CysLT1 before and after desensitization with topical lysine aspirin

Sousa AR et al. New Engl J Med 347:1499, 2002

Leukotriene Modifiers in AERD

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

Why So Much CysLTs?

•  also, why so many CysLT receptors?

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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<0.001 <0.001 <0.001

5 10 15 >20

eosinophils/400x field

Payne, S., et al. Laryngoscope 2011

AERD Associated With Eosinophilic Inflammation:

Cowburn AS, et al. J Clin Invest 1998;101:834.

Immunostaining for ECP (EG2+ eosinophils) in bronchial mucosal from patients with AERD, ATA, and normal subjects:

And, II: Too many eosinophils expressing LTC4S

Relative expression of CysLTs and LTC4S mRNA"

Pérez-Novo, CA et al. J. Allergy Clin. Immunol. 2005;115:1189-96.

Nl NES CHES AERD Nl NES CHES AERD

# of cells staining for LTC4S in bronchial mucosal biopsies from patients with AERD, ATA and normal subjects

Cowburn AS, et al. J Clin Invest 1998;101:834.

Proportion of LTC4 synthase+ cells colocalizing to eosinophils (EG2+), mast cells (AA1+), and macrophages (CD68+)

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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)

2ΔCT

x1

0-2 (I

L-4,

IL-1

3); x

10-4

(IFN

-γ);

x10-5

(IL-

5)

0

20

10

40

30

50

IL-4 IL-5 IL-13 IFN-γ 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

-γ 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

-γ A

PC

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

Eosinophil Intracellular Cytokine Expression  

CHES AERD IL-4 33.2±8.8 34.8±10.1 IFN-γ 15.0±3.4 28.2±10.7

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

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i  

i  

isotype   IFN-­‐γ

IFN-γ Immunofluorescence: CHES

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

IFN-γ Immunofluorescence: AERD

i  

i  

isotype   IFN-­‐γ

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-­‐γ  Poten=a=on  of  Eosinophilopoiesis  

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

+ IFN-γ

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

Eosinophil cellSamples_E-IFNy.fcsEvent Count: 7870

0 102 103 104 105

<APC-A>: Siglec 8

0

102

103

104

105

<PE-

A>: C

CR

3

5.81 4.19

4.3185.7

Eosinophil cellSamples_E+IFNy.fcsEvent Count: 7907

0 102 103 104 105

<APC-A>: Siglec8

0

102

103

104

105

<PE-

A>: C

CR

3

1.83 8.56

3.6585.9

Eosinophil cellSamples_M-IFNy.fcsEvent Count: 6143

0 102 103 104 105

<APC-A>

0

102

103

104

105

<PE-

A>

1.79 7.5

11.379.4

Eosinophil cellSamples_M+IFNy.fcsEvent Count: 4088

0 102 103 104 105

<APC-A>

0

102

103

104

105

<PE-

A>

1.44 19

9.1570.5

-IFN-γ +IFN-γ

Siglec 8 Siglec 8

CCR3

CCR3

Enhanced eosinophilopoiesis in additional presence of IFN-γ:

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

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Enhanced  Eosinophilopoiesis  by  IFN-­‐γ

0

5

10

15

20

25

30

35

Sigl

ec8+ /

CCR3

+ (%

)

-IFN-γ +IFN-γ

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

IFN-γ Activation of Eosinophilopoiesis  

Siglec8 ECP CysLT1 CysLT2 P2Y12 EP2 LTC4S Cox2 PgD2S -IFN-γ .0063±

.0017 .109± .030

.052±

.024 .00059± .00031

.0068±

.0021 .072± .032

.033±

.014 .0043± .0013

.059±

.023 +IFN-γ .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 (EF1α) (2∆∆CT) (n=12)

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

*p<0.05 compared to without IFN-γ

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

[EDN

]  ng/10

5  eosinop

hils  

EDN  Produc=on  

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

CysLT  Produc=on  

[Cys

LTs]

pg/

ml

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

What About the CysLT Receptor Over-Expression?

CysLT1 Receptor Expression on Nasal Mucosal Inflammatory Cells in AERD

Sousa AR et al. New Engl J Med 347:1499, 2002 Immunoreactive cells were counted in specimens of nasal submucosa

ATA AERD

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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-γ

81 kd

38.5 kd

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

Why don’t 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<0.03

mM

Aspirin/Salicylate Activation of Eosinophils

*but again, no obvious difference between aspirin tolerant and AERD

and, surprisingly, modest CysLT production

Why is aspirin activation happening with control eosinophils and how is AERD different? •  More eosinophils in AERD •  Eosinophil activation may occur in aspirin tolerant

asthma, but PgE2 acting ELSEWHERE is protective •  More LTC4S in tissue eosinophils in AERD •  PgE2 primarily acts to block CysLT production ���

(5-LO activity) •  It is OK to activate eosinophils and release CysLTs

as long as LTE4R is not simultaneously over-expressed

Hawley SA et al. Science 2012;336:918-22 Din FVN et al. Gastroenterology 2012;142:1504-15

OR…

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EDN  (ng/10

5  eosinophils)  

-­‐IFN-­‐γ +IFN-­‐γ Medium   Medium  LysASA   LysASA  PMA/ion  PMA/ion  

1  mM  1  mM   10  mM  10  mM  

*  

*  

*  

*  

*p<0.05

EDN  Produc=on  

CysLT  (pg/10

5  eosinophils)  

-­‐IFN-­‐γ +IFN-­‐γ Medium   Medium  LysASA   LysASA  PMA/

ion  PMA/ion  

1  mM  1  mM   10  mM  10  mM  1  mM   1  mM  10  mM   10  mM  

Ketorlac   Ketorlac  

*  *  

**  

**  

**  

*  

**  

*p<0.05; **p<0.001

CysLT  Produc=on  

Conclusions •  AERD is a disease of over-expression of LTC4 synthase,

increased production of CysLTs, and increased expression of CysLT receptors

•  AERD reflects over-expression of Th2 (IL-4/IL-13) and Th1 (IFN-γ)-associated cytokines

•  IFN-γ increases expression of LTC4S in newly developing eosinophils

•  IFN-γ increases expression of CysLT receptors •  Aspirin can directly activate eosinophils (and mast cells)

-  And this includes CysLTs if those eosinophils were allowed to mature in a “high” IFN-γ milieu

-  IFN-γ will contribute to a milieu in which allergic sensitization (atopy) might not develop