mechanisms behind the cf - abpa link 1. cf – abpa linkspulmonary hypersensitivity disease mediated...

1

Mechanisms behind the CF - ABPA link

Dr. Dominik Hartl

Cystic Fibrosis Center

University of Munich

1. CF – ABPA links

2. The role of chemokines


ABPA is a Th2 disease

Is CF a Th2 disease?

ABPA


ABPA

2

Allergic Bronchopulmonary Aspergillosis (ABPA)

Pulmonary hypersensitivity disease mediated by an allergic responseto Aspergillus fumigatus.



Occurs in patients with impaired pulmonary immunity (asthma, CF)CF about 10-20% of CF patients (mainly > 15 years)




Severe consequences: wheezing, decline of lung function, fibrosis




Severe consequences: wheezing, decline of lung function, fibrosis

Clinical need for reliable serum markers of ABPA (Ste vens et al., 2003)

3



ABPA


Majority of T cell clones CD4+ IL-4+ cells(Knutsen 1989, 2003; Chauhan 1996)



IL-4 and IL-5 increased in BALF(Walker 1994)




A. fumigatus antigen-specific T-cell lines secrete abundant levels of IL-4 in an autocrine fashion (Knutsen 1994)

4




A. fumigatus antigen-specific T-cell lines secrete abundant levels of IL-4 in an autocrine fashion (Knutsen 1994)

PBMCs from ABPA patients: increased sensitivity to IL-4 with upregulation of the low-affinity IgE receptor on B cells(Knutsen 2003, Khan 2000, McClellan 1999)



Is CF a Th2 disease?ABPA in CF / ABPA in asthma

ABPA

Is CF a Th2 disease?CF PBMCs: PMA/TCR => reduced IFN- γ secretion and increased IL-10 mRNAexpression (Moss 2000)


CD4+ T cells from CFTR-/- mice: # TCR activation: IL-4 production ↑, NFAT ↑ compared to WT # A. fumigatus = IL-4 in BALF ↑ IgE and IgG1 in serum ↑

(Allard 2006, Müller 2006)

5




CFTR-/- epithelial cells: RANTES (Th1 chemokine) ↓(Schwiebert 1999). Impaired NOS2 / STAT1 signaling (Kelley 2000)





Whole blood cell cultures stimulated with LPS/PHA:IL-4, IL-10 and IFN-γ secretion => "low responders" and „high responders“; Pseudomonas infection => higher IL-4 levels(Brazova J, 2005)






Pseudomonas infection results in pulmonary Th2 responseBALF: CCR4+CD4+ Th2 cells; IL-4, IL-13 and TARC ↑(Hartl 2006)






Pseudomonas infection results in pulmonary Th2 responseBALF: CCR4+CD4+ Th2 cells; IL-4, IL-13 and TARC ↑(Hartl 2006)

Due

to P

seu

do

mo

nas

?

6

CF – ABPA links

CFTR: Intrinsic/systemic Th2 bias

CF – ABPA links

CFTR: Intrinsic/systemic Th2 biasPseudomonas: Pulmonary Th2 shift

CF – ABPA links


Intensified DC-antigen contact due to impaired ASL (Dubin et al. 2007)

CF – ABPA links


Intensified DC-antigen contact due to impaired ASL (Dubin et al. 2007)

CF: Pulmonary microenvironment

susceptible towards Th2 responses

7

Chemokines

Chemokines = chemo tactic cyto kines

Chemokines

Rolling Activation Adhesion Diapedesis

4000µm/sec40µm/sec seconds minutes ~10 minutes

Lung TissueChemokines

(e.g.“Th1“ or „Th2“)

Chemokines

Chemokines = chemo tactic cyto kines

Th1/Th2 Chemokines = Th1/Th2 Cytokines

Th1Th1Th1Th1

Th2Th2Th2Th2

RANTES,RANTES,RANTES,RANTES,ITAC, IPITAC, IPITAC, IPITAC, IP----10101010 TARC, MDCTARC, MDCTARC, MDCTARC, MDC

IIII----309309309309

Sallusto et al., Annu Rev Immunol. 2000

Th1/Th2 Chemokines

CCR4

8

Chemokines in CF lung disease

Predominant chemokine: IL-8 (KC, MIP-2)

Lack of Th1 chemokine RANTES secretion in CFTR-/- epithelial cells (Schwiebert et al. 1999)

High levels of Th2 chemokines TARC and MDC and in CF BALF, especially Pseudomonas+ (Hartl et al., 2006)

Hartl et al., JACI, 2006


BAL

Hartl et al., JACI, 2006


BAL



Lack of RANTES (Th1 chemokine) secretion in CFTR-/- epithelial cells (Schwiebert et al., 1999 )

ABPA:TARC/CCR4 plays a critical role in murine models of ABPA(Schuh & Hogaboam, 2002)


9

CCR4+/+0

5

10

15

20

25

Air

way

Res

ista

nce

(cm

H2O

/ml/s

ec)

Schuh J, Hogaboam CM., FASEB 2002

Allergic Aspergillosis

CCR4+/+ CCR4+/+ CCR4-/-0

5

10

15

20

25

Air

way

Res

ista

nce

(cm

H2O

/ml/s

ec)

Schuh J, Hogaboam CM., FASEB 2002

Allergic Aspergillosis

TARC/CCR4 signalling plays a critical role in murine allergic aspergillosis

CCR4+/+ CCR4-/-

Th1

/Th2

dys

bala

nce

in C

FA

spe

cial

rol

e fo

r T

AR

C?



Lack of Th1 chemokine RANTES secretion in CFTR-/- epithelial cells (Schwiebert et al., 1999 )

ABPA:TARC/CCR4 plays a critical role in murine models of ABPA(Schuh & Hogaboam, 2002)


TNF-ααααIL-1β β β β

Th2cell

TARC/MDC

CCR4

Th2

IL-4

AlAlV

V

10

TNF-ααααIL-1β β β β

Th2cell

TARC/MDC

CCR4

Th2

IL-4

AlAlV

V

TARC

ABPA in CF is characterized by a strong Th2 respons e

Hypothesis generation


Animal models suggest a critical role for TARCin allergic aspergillosis




TARC (CCL17) is consistently detectable in human se rum


11




TARC (CCL17) is consistently detectable in human se rum

TARC may be a serum marker for ABPA

Is TARC useful to differentiate CF ABPA patients from

• CF patients colonized with Aspergillus fumigatus

• CF patients sensitized to Aspergillus fumigatus

• Atopic CF patients

• Atopic healthy control subjects

n.d.5/5/23/6/44/2n.d.9/6/1 dF508homo/hetero/other

0/133/123/130/60/67/16S.maltophilia

0/136/129/132/60/610/16P. aeruginosa

0/130/123/13*0/64/6*10/16**Itraconazole

0/131/121/130/64/6*11/16**Systemic steroids

5/137/127/133/66/613/16Inhaled steroids

n.d.26 (17-40)24 (15-41)29 (19-37)31 (17-35)22 (12-39)MEF25-75 (% pred)

n.d.72 (41-93)65 (36-91)71 (45-84)83 (65-95)61 (34-89)FVC (% of pred)

n.d.61 (37-92)57 (15-104)60 (24-93)62 (41-79)46 (32-81)FEV1 (% of pred)

8 (2-10)*7 (4-11) *2 (0-5) 5 (0-8) 9 (3-18)*10 (5-20)*Blood Eosinophils(%)

0/130/1213/13**6/6**6/6**16/16**A. fumigatus in sputum culture

n.d.n.d.0 (0-0)0.36 (0-5.2)1.7 (0-7.1)2.4 (0.1-8.3)rAsp f6 (kU/l)

n.d.n.d.0 (0-0)0.38 (0-4.9)10.5 (1.2-34.5)13.8 (0.2-53.4)rAsp f4 (kU/l)

0 (0-0)0 (0-0)0 (0-0)3 (0-4)**4 (0-5)**4 (0-6)**Specific IgE to A. fumigatus

838 (446-4165)*627 (338-2395)*47 (1-120) 643 (395-988) 1899 (1321-2242)*3254 (445-6606)**IgE (IU/ml)

00.8 (0-2)0.6 (0-1.7) 0.4 (0-1.6) 0.3 (0-1.2)0.6 (0-6.5)CRP (mg/dl)

6/77/56/7 3/3 3/310/6Sex (M/F)

17 (7-19)21 (14-37)24 (11-42) 21 (15-28) 24 (21-30)18 (11-31)§Age (years)

1312136616Number

Non-CFatopic

CF atopic

CF A. fumigatuscolonization

CF A. fumigatussensitization

Asthma ABPA

CF ABPA

Hartl et al., Am J Respir Crit Care Med 2006

Cytokines

12

1

10

100

1000

CF ABPA

Atopic controls

IL-4 IL-5 IL-13 IFN-γγγγ IL-12

CF atopic

Th2 Th1

Asthma ABPA

CF Af-sensitized

CF Af-colonized

**

**

**

pg/m

l


Cytokines


TARC serum levels are increased in ABPA patients

CF

AB

PA

-IgE

pea

k

CF

AB

PA

-ave

rage

Ast

hma

AB

PA

-IgE

pea

k

CF

Af-

sens

itiz

ed

CF

Af-

colo

nize

d

CF

ato

pic

Ato

pic

cont

rols

0

500

1000

1500

2000

2500

3000

***

**

****

TA

RC

(pg

/ml)



0

250

500

750

1000

1250

1500

1750

2000

2250

2500

2750 **

Corticosteroids + -

TAR

C (

pg/m

l)



13

Conclusions Study I

1. TARC serum levels indicate clinically active ABPA in CF and asthma patients vs Aspergillus colonizationand sensitization

2. TARC serum levels associated with corticosteroidtreatment


Open Questions:

1. Can these results be reproduced in a different CF population? CF populations and centers show a great variability in their microbiological colonization, their atopic status and their genetic background

2. Is the elevation of TARC levels just an epi-phenomenon of the allergy in ABPA patients?

3. What is the diagnostic accuracy of TARC compared to other serological markers for ABPA?

CF

2544509781050IgE (IU/ml)

9/7 4/46/67/5Sex (M/F)

37 (24-78)13 (0-72)54 (0-61)40 (25-80)Duration of continuous serum samples (months)

85316287Number of serum samples:

13 (6-29) 17 (9-40) 12 (7-42)12 (5-20)Age at first serum sample in years

1681212Number of patients

controlrAsp f4/f6IgE

Non-ABPAABPA

ABPA study II: Switzerland

Latzin et al., ERJ 2007132-389106-317154-531243-956TARC level range [pg/ml]

177-281167-245213-342465-673TARC level IQR [pg/ml]

207 200256589TARC level median [pg/ml]

36235485Number of measurements

1681212Number of patients

Controlf4/f6IgE

Non-ABPA groupsABPA group

ABPA IgE f4/f6 control0

100

200

300

400

500

600

700

TA

RC

pg/

ml

1. Can these results be confirmed in a different CF

population?

p<0.01

p<0.01

p<0.01

Latzin et al., ERJ 2007

14

2. Is the elevation of TARC levels specific for ABP A or anepi-phenomena of the elevated IgE-levels in ABPApatients?


clincal ABPA no clinical ABPA

seru

m T

AR

C le

vels

[pg/

mL]

0

200

400

600

800

serum IgE

levels [IU/m

L]

0

1000

2000

TARCIgE

2. Is the elevation of TARC levels specific for ABP A or anepi-phenomena of the elevated IgE-levels in ABPApatients?

Latzin et al., ERJ 2007Nested matched case-control: n=10

clincal ABPA no clinical ABPA

seru

m T

AR

C le

vels

[pg/

mL]

0

200

400

600

800

serum rA

sp levels [IU/m

L]

0

20

40

60

80

100TARCrAsp f4

rAsp f6

2. Is the elevation of TARC levels specific for ABP A or anepi-phenomena of the elevated rAsp f4/f6 in ABPApatients?

Latzin et al., ERJ 2007Nested matched case-control: n=10

3. What is the diagnostic accuracy of TARC compared toother serological markers for ABPA?


15

0.730.800.790.820.830.840.98Area under the ROC

0.550.410.260.400.380.440.09Negative Likelihood ratio

3.34.82.84.74.13.417.3Positive Likelihood ratio

73.279.074.879.078.174.393.4Diagnostic accuracy [%]

83.786.571.285.983.381.091.2Specificity [%]

53.764.681.765.968.364.789Sensitivity [%]

IgGrAsp

f6rAsp

f4rAsp

f3rAsp

f1IgETARC

Cut-off levels of 386 pg/ml for TARC (a cut-off level of 398 pg/ml yields to the same diagnostic accuracy with a sensitivity of 89.4% and a specificity of 96.5%, respectively), 514 IU/ml for IgE, 75 IU/ml for rAsp f1, 140 IU/ml for r Asp f3, 10 IU/ml for r Asp f4, 16 IU/ml for r Asp f6 and 140 EU/ml for IgG.



0.730.800.790.820.830.840.98Area under the ROC

0.550.410.260.400.380.440.09Negative Likelihood ratio

3.34.82.84.74.13.417.3Positive Likelihood ratio

73.279.074.879.078.174.393.4Diagnostic accuracy [%]

83.786.571.285.983.381.091.2Specificity [%]

53.764.681.765.968.364.789Sensitivity [%]

IgGrAsp

f6rAsp

f4rAsp

f3rAsp

f1IgETARC

Cut-off levels of 386 pg/ml for TARC (a cut-off level of 398 pg/ml yields to the same diagnostic accuracy with a sensitivity of 89.4% and a specificity of 96.5%, respectively), 514 IU/ml for IgE, 75 IU/ml for rAsp f1, 140 IU/ml for r Asp f3, 10 IU/ml for r Asp f4, 16 IU/ml for r Asp f6 and 140 EU/ml for IgG.




0.0

00

.25

0.5

00

.75

1.0

0S

ensi

tivity

0.00 0.25 0.50 0.75 1.001-Specificity

ROC curves for TARC, total IgE, rAsp f4 and rAsp f6 including all serum samples.

TARC

IgE rAspf4

rAspf6


• Cut-off levels of 386 pg/ml for TARC• including all available time points (n=265)


In CF patients who develop ABPA TARC serum levels are early elevated (>400pg/ml)

Longitudinal analysis

16

Methodological issues

Immuno- sandwich ELISA (R&D Systems, MN, USA)

Pitfalls:>> sensitive to pre-analytical handling (freezing, thawing, blood in serum)>> reliable standard curves>> detection limits (~7 pg/ml - 3000 pg/ml) >> Intra-assay variability (5 serum samples 10x same assay): CV = 6 - 9.3%. >> Inter-assay variability (5 serum samples 5 consecutive assay): CV = 8.4 - 17%

=> Multicenter study => One center, one technician, one thawing

Hartl D and Hogaboam CM, Allergy & Inflammation 2006

Hartl D and Hogaboam CM, Allergy & Inflammation 2006

TSLP+

IDO, kynurenine

Th17

Open questions: CF – ABPA link

Th2 imbalance in CF• Basic mechanism: CFTR => Th2 (NFAT, Ca)?

• Subgroup of patients (high/low responders), SNPs?

• Age, disease severity, CFTR mutation, Pseudomonas associated?

Immunological key players• TARC (predictive value? SNPs? effects on pulmonary cells?)

• Role of IL-17/IL-23: interface between T cells and PMNs (Dubin and Kolls , 2007)

• Role of DCs / regulatory T cells (Romani L)

17

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements

MunichMunichMunichMunichDietrich Reinhardt

Matthias Griese

Markus Krane

DFG Grant

BernPhilipp Latzin

Carmen Casaulta

Martin Schöni

Urs Frey

Cory Hogaboam (Ann Arbor)

Dirk Roos (Amsterdam)

Taco Kuijpers (Amsterdam)

Gernot Zissel (Freiburg)

Richard Moss (Stanford)

mechanisms behind the cf - abpa link 1. cf – abpa linkspulmonary hypersensitivity disease mediated...

Documents