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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
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