Cracking the Fungal Armor -Studies on Host Defense Mechanisms

agains A. fumigatus

Tobias M. Hohl, MD, PhDMemorial Sloan-Kettering Cancer Center

hohlt@mskcc org orghohlt@mskcc.org.org

A. fumigatus Germinationg

Intact pulmonary

Conidial clearance

Intact pulmonaryimmune defense

Defective pulmonaryDefective pulmonaryimmune defense

Tissue-invasive hyphae

Host Immune DefenseHost Immune Defense against A. fumigatus

• Recognition of inhaled spores by the innate immune systemy

• Modulation of inflammatory yresponses by antifungal therapy

• Monocytes and the initiation of CD4 T cell responsesp

Live Conidia induce Airway NeutrophilLive Conidia induce Airway Neutrophil Recruitment

36

42Total CellsMacrophagesNeutrophils

intratracheal

24

30

Neutrophils

12

18

6

Vehicle Heat-killed Live ConidiaResting conidia

Hohl, T.M. et al., PloS Pathog. 1:e30, 2005.

Live Conidia induce TNF/ CXCL2 SecretionLive Conidia induce TNF/ CXCL2 Secretion by Alveolar Macrophages

TNF CXCL2

18

24

45

60

6

12

15

30

ng/m

l

Live HK Medium LPSLive HK Medium LPS

Hohl, T.M. et al., PloS Pathog. 1:e30, 2005.

Killed Germinating Conidia are highly Inflammatoryg g y y

Conidial Swelling Germ Tube Formation

t = 0 3 h 5 h 7 h

9TNFCXCL2

3

6

ng/m

l

3 h 5 h 7 h

Heat-killed

Hohl, T.M. et al., PloS Pathog. 1:e30, 2005.

Killed swollen Conidia induce NeutrophilKilled swollen Conidia induce Neutrophil Influx into the BAL fluid

Total Cells30

MacrophagesNeutrophils

18

24

12

6

LiveHeat-killedConidia

Heat killedswollen conidia

Hohl, T.M. et al., PloS Pathog. 1:e30, 2005.

Swollen Conidia and GermlingsSwollen Conidia and Germlings expose β-glucan on their surface

Anti β-glucan Isotype Control AbAnti β-glucan Isotype Control Ab

Hohl, T.M. et al., PloS Pathog. 1:e30, 2005.

Dectin-1 binds and signals i t β (1 3) lin response to β-(1,3) glucan

Brown, G. D., Nat Rev Immunol 6:33-43, 2006.

Conidia Stimulate Dectin 1 and MyD88Conidia Stimulate Dectin-1- and MyD88-dependent Pathways

CXCL2TNF

2

3

/ml 2

3

1

ng/

1

WT MyD88-/-MyD88-/-WT

anti-Dectin - + - + - + - +

Hohl, T.M. et al., PloS Pathog. 1:e30, 2005.

Host Immune DefenseHost Immune Defense against A. fumigatus

• Recognition of inhaled spores by the innate immune systemy

• Modulation of inflammatory yresponses by antifungal therapy

• Monocytes and the initiation of CD4 T cell responsesp

Modulation of Host Inflammatory ResponsesModulation of Host Inflammatory Responses by Antifungal Therapy

• Echinocandins target fungal-β-D-glucan synthase• Echinocandins reduce A. fumigatus bulk β-glucan levels

(Kahn, J. et al., Antimicrob Agents Chemother 50:2214-2216, 2006)E hi di d t f ll i hibit A f i t th t i d• Echinocandins do not fully inhibit A. fumigatus growth, yet induce prominent morphologic changes at or above the MEC

1 x MEC Caspofungin (63 ng/ml) No Caspofungin

Caspofungin Decreases Macrophage Inflammatory Responses to ConidiaResponses to Conidia

1.2

1.8

ng/m

l)

No Caspofungin

0.6TNF

(n

**

* *No Caspofungin

Caspo (ng/ml) 0 4 8 16 31 63 125 250500

Caspofungin (500 ng/ml)

BMMφ TNF/CXCL2 release (500 ng/ml caspofungin vs. no drug exposure):

• TNF 0.49 ± 0.04* (range 0.46-0.54; n=4)• CXCL2 0.55 ± 0.10* (range 0.43-0.62; n=4)

Hohl, T.M. et al. J Infect Dis, 2008.

Caspofungin Enhances Macrophage InflammatoryCaspofungin Enhances Macrophage Inflammatory Responses to Hyphae

8

10

6

8

ml)

g/m

l)* * * * * * * *

2

4

6

2

4

TNF

(ng/

m

CXC

L2 (n

gC

0 4 8 16 31 63 125 250 500 0 4 8 16 31 63 125 250 500Caspo (ng/ml)

BMMφ TNF/CXCL2 release (500 ng/ml caspofungin vs. no drug exposure) • TNF 4.11 ± 2.39* (range 1.90-7.84; n=8)• CXCL2 2.90 ± 1.40* (range 1.53-5.41; n=8)

Hohl, T.M. et al. J Infect Dis, 2008.

Caspofungin Modulates Dectin-1-dependent Inflammatory Responses to Conidia Germlings and HyphaeResponses to Conidia, Germlings, and Hyphae

67

HyphaeConidia1.2 3.5

3

Germlings

23456

0 4

0.8

NF

(ng/

ml)

11.52

2.53

12

Caspofungin

0.4TN

+-

0.51

+- +-

Dectin-1-dependent TNF release

Dectin-1-independent TNF release

Hohl, T.M. et al. J Infect Dis, 2008.

Effects of Echinocandin Drugs on β-glucan ExposureEffects of Echinocandin Drugs on β-glucan Exposure

No Caspofungin

DIC anti-β-glucanDIC

Caspofungin

Aanti-β-glucan

8 h

10 h

Effects of Echinocandin Drugs on β-glucan Exposure

No CaspofunginCaspofungin No Caspofungin

DIC anti-β-glucanDIC

Caspofungin

Aanti-β-glucan

12 h

15 h

18 h

Hohl, T.M. et al. J Infect Dis, 2008.

Q tit ti A l i f β l I ti it i t dQuantitative Analysis of β-glucan Immunoreactivity associated with Caspofungin-treated and Untreated Hyphae

Integrated Fluorescence Intensity/Fungal Mass(Arbitrary Units)

Caspofungin-treated Hyphae Untreated Hyphae

Expt. 1 21.4 ± 8.3* 1.83 ± 0.73

Expt. 2 43.7 ± 7.0* 2.96 ± 4.67

Each value represents the average ratio (± SD) of β-glucan immunofluorescence intensity normalized to hyphal mass as calculated from 4-5 fields of view per condition.

* p <0.02 compared to control condition (untreated hyphae).

Hohl, T.M. et al. J Infect Dis, 2008.

Echinocandin Drugs have an Immunopharmacologic Mechanism of

Action

Host Immune DefenseHost Immune Defense against A. fumigatus

• Recognition of inhaled spores by the innate immune systemy

• Modulation of inflammatory yresponses by antifungal therapy

• Monocytes and the initiation of CD4 T cell responsesp

Monocyte-derived Populations in HostMonocyte-derived Populations in Host Defense against A. fumigatus

Development of an ExperimentalDevelopment of an Experimental System to Track and Ablate Monocytes

Recruitment of GFP+ monocytes and myeloid DCs into the lungs of A. fumigatus-infected mice

14.6

A. fumigatusg g

31.4

MDCs (2.7 M)

60.6 Mo (5.2 M)

29.17.2

Uninfected

48D11

c

GFP

MDCs(0.4 M)

Mo (0 7 M)48CD

CD11b

G

Ly6G

Mo (0.7 M)

Cell Recruitment to mLN after intratracheali f ti ith A f i t idiinfection with A. fumigatus conidia

A. fumigatus Uninfected

2.24 M 4.16 M 0.52 M 0.25 M

5 1 09 5 1 17 5 0 21 5 0 04

103

104

105

GFP

1.09

103

104

105 1.17

103

104

105 0.21

103

104

105 0.04

0 102 103 104 105

CD11b

0102

0 102 103 104 105

0102

0 102 103 104 105

0102

0 102 103 104 105

0102

30

40

50InfectedUninfected

GFP

+ce

lls03

cells

)

10

20

CD

11b+

(x 1

0

Characterization of GFP+ cells in the mLN48 h t i f ti ith A f i t48 h post-infection with A. fumigatus

1.17

1.17 Mo

P

R249.7

38Mo

MDC

11b

MDC

GFP

CD11b

49.7

CD

CD11c MDCCD11b CD11c

Class II CD86 Ly6C

CD11b+CD11c+GFP+ cells transportCD11b CD11c GFP cells transport labelled conidia to mLN

10 4

10 5

P

0.4

15

20

25

2

2.5

100

150

51.8

0

10 2

10 3GFP

0

5

10

15

# C

ells

2.28

0

0.5

1

1.5

# C

ells

95.2

0

50

100

# C

ells

48.2

200

0 10 2 10 3 10 4 10 5

CD11b

10 51.4

0 10 2 10 3 10 4 10 5

AF633-Conidia

0

20

25

0 10 3 10 4 10 5

CD11c

00 10 3 10 4 10 5

CD11c

0

50

100

150

# C

ells

52.2

47.8

10 2

10 3

10 4

GFP

5

10

15

20#

Cel

ls

0.018

0 10 3 10 4 10 5

CD11c

00 10 2 10 3 10 4 10 5

CD11b

0

0 10 2 10 3 10 4 10 5

Conidia

0

5

Depletion of CCR2-expressing cellsDepletion of CCR2-expressing cells reduces conidial trafficking to the mLN

Ablation of Lung DC subsets in CCR2 Depleter

CCR2 Depleter mice cannot prime A.

f i t ifi CD4 T llfumigatus-specific CD4 T cell responsesCCR2 depleter orC57BL/6 (Thy 1.2) Infection via i.t. route

AF-specific CD4 T cells (Thy 1.1/1.2)

mLN CD4 Gate

+6-1 0 +1

0.06

0.09

0.12

Cells

103

104

105

hy 1

.1

0.15

CCR2 Depleter(DT t t d)

mLN CD4 GateDT DT

0 103 104 105

CFSE

0

0.03

#

0 102 103 104 105

Thy 1.2

0

102

10

Th(DT-treated)

6

9

12

Cells

103

104

105

y 1.

1

3.66

Non-Tg Control(DT t t d)

0 103 104 105

CFSE

0

3

6

#

0 102 103 104 105

Thy 1.2

0

102

10

Th(DT-treated)

Summary

• CCR2 reporter and depleter mice represent valuable tools to dissect the role of monocytes and monocyte derived cells in microbial defenseand monocyte-derived cells in microbial defense

• Monocyte-derived lung DCs (CD11b+) transport conidia to draining lymph nodesg y p

• Ablation results in loss of A. fumigatus-specific CD4 T cell primingM t i t ib t t i i d A• Monocytopenia may contribute to impaired A. fumigatus CD4 T cell responses in patients undergoing HSCTg g

AcknowledgementsAcknowledgements

MSKCC Albert Einstein College of Medicine• Eric Pamer• Heather Van Epps• Amariliz Rivera• Monica Mircescu

• Marta Feldmesser• Patrick Chen

P bli H lth R h I tit t• Alena Gallegos• Katharina Brandl• Ting Jia• Natalya Serbina

Public Health Research Institute, Newark, NJ

• David Perlin

• Natalya Serbina

• Alexander Lesokhin• Alan Houghton

University of Cape Town• Gordon Brown

• Mabel Ryder• James Fagin

Research Support: Charles F. Revson Foundation, NIH K08 Award