Cracking the Fungal Armor - Studies on Host Defense Mechanisms - - PowerPoint PPT Presentation

▶
cracking the fungal armor studies on host defense
SMART_READER_LITE
LIVE PREVIEW

Cracking the Fungal Armor - Studies on Host Defense Mechanisms - - PowerPoint PPT Presentation

Dec 01, 2023 32 likes •357 views

Cracking the Fungal Armor - Studies on Host Defense Mechanisms agains A. fumigatus Tobias M. Hohl, MD, PhD Memorial Sloan-Kettering Cancer Center hohlt@mskcc org org hohlt@mskcc.org .org A. fumigatus Germination g Intact pulmonary Intact

slide-1
SLIDE 1

Cracking the Fungal Armor - Studies on Host Defense Mechanisms agains A. fumigatus

Tobias M. Hohl, MD, PhD Memorial Sloan-Kettering Cancer Center hohlt@mskcc org org hohlt@mskcc.org.org

slide-2
SLIDE 2
  • A. fumigatus Germination

g

Intact pulmonary Conidial clearance Intact pulmonary immune defense Defective pulmonary Defective pulmonary immune defense Tissue-invasive hyphae

slide-3
SLIDE 3

Host Immune Defense Host Immune Defense against A. fumigatus

  • Recognition of inhaled spores by the

innate immune system y

  • Modulation of inflammatory

y responses by antifungal therapy

  • Monocytes and the initiation of CD4

T cell responses p

slide-4
SLIDE 4

Live Conidia induce Airway Neutrophil Live Conidia induce Airway Neutrophil Recruitment

36 42

Total Cells Macrophages Neutrophils

intratracheal

24 30

Neutrophils

12 18 6 Vehicle Heat-killed Live Conidia Resting conidia Hohl, T.M. et al., PloS Pathog. 1:e30, 2005.

slide-5
SLIDE 5

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

TNF CXCL2

18 24 45 60 6 12 15 30

ng/ml

Live HK Medium LPS Live HK Medium LPS

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

slide-6
SLIDE 6

Killed Germinating Conidia are highly Inflammatory g g y y

Conidial Swelling Germ Tube Formation

t = 0 3 h 5 h 7 h 9 TNF CXCL2 3 6 ng/ml 3 h 5 h 7 h Heat-killed Hohl, T.M. et al., PloS Pathog. 1:e30, 2005.

slide-7
SLIDE 7

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

Total Cells

30

Macrophages Neutrophils

18 24 12 6 Live Heat-killed Conidia Heat killed swollen conidia Hohl, T.M. et al., PloS Pathog. 1:e30, 2005.

slide-8
SLIDE 8

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

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

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

slide-9
SLIDE 9

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

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

slide-10
SLIDE 10

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

CXCL2 TNF

2 3 /ml 2 3 1 ng/ 1 WT MyD88-/- MyD88-/- WT anti-Dectin

  • +
  • +
  • +
  • +

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

slide-11
SLIDE 11
slide-12
SLIDE 12

Host Immune Defense Host Immune Defense against A. fumigatus

  • Recognition of inhaled spores by the

innate immune system y

  • Modulation of inflammatory

y responses by antifungal therapy

  • Monocytes and the initiation of CD4

T cell responses p

slide-13
SLIDE 13

Modulation of Host Inflammatory Responses Modulation 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

slide-14
SLIDE 14

Caspofungin Decreases Macrophage Inflammatory Responses to Conidia Responses to Conidia

1.2 1.8 ng/ml)

No Caspofungin

0.6 TNF (n * * * *

No Caspofungin

Caspo (ng/ml) 4 8 16 31 63 125250500

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.

slide-15
SLIDE 15

Caspofungin Enhances Macrophage Inflammatory Caspofungin Enhances Macrophage Inflammatory Responses to Hyphae

8 10 6 8 ml) g/ml) * * * * * * * * 2 4 6 2 4 TNF (ng/m CXCL2 (ng C 4 8 16 31 63 125 250 500 4 8 16 31 63 125 250 500 Caspo (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.

slide-16
SLIDE 16

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

6 7

Hyphae Conidia

1.2 3.5 3

Germlings

2 3 4 5 6 0 4 0.8 NF (ng/ml) 1 1.5 2 2.5 3 1 2 Caspofungin 0.4 TN +

  • 0.5

1 +

  • +
  • Dectin-1-dependent TNF release

Dectin-1-independent TNF release

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

slide-17
SLIDE 17

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

No Caspofungin DIC anti-β-glucan DIC Caspofungin

A

anti-β-glucan 8 h 10 h

slide-18
SLIDE 18

Effects of Echinocandin Drugs on β-glucan Exposure

No Caspofungin Caspofungin No Caspofungin DIC anti-β-glucan DIC Caspofungin

A

anti-β-glucan 12 h 15 h 18 h

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

slide-19
SLIDE 19

Q tit ti A l i f β l I ti it i t d Quantitative 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.

slide-20
SLIDE 20

Echinocandin Drugs have an Immunopharmacologic Mechanism of Action

slide-21
SLIDE 21

Host Immune Defense Host Immune Defense against A. fumigatus

  • Recognition of inhaled spores by the

innate immune system y

  • Modulation of inflammatory

y responses by antifungal therapy

  • Monocytes and the initiation of CD4

T cell responses p

slide-22
SLIDE 22

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

slide-23
SLIDE 23

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

slide-24
SLIDE 24

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

14.6

  • A. fumigatus

g g

31.4

MDCs (2.7 M)

60.6

Mo (5.2 M)

29.1 7.2

Uninfected

48

D11c GFP

MDCs(0.4 M) Mo (0 7 M)

48

CD CD11b G Ly6G

Mo (0.7 M)

slide-25
SLIDE 25

Cell Recruitment to mLN after intratracheal i f ti ith A f i t idi infection 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 10 3 10 4 10 5

GFP

1.09 10 3 10 4 10 5 1.17 10 3 10 4 10 5 0.21 10 3 10 4 10 5 0.04 0 10 2 10 3 10 4 10 5

CD11b

10 2 0 10 2 10 3 10 4 10 5 10 2 0 10 2 10 3 10 4 10 5 10 2 0 10 2 10 3 10 4 10 5 10 2

30 40 50 Infected Uninfected

GFP+ cells 03 cells)

10 20

CD11b+ (x 10

slide-26
SLIDE 26

Characterization of GFP+ cells in the mLN 48 h t i f ti ith A f i t 48 h post-infection with A. fumigatus

1.17

1.17

Mo

P

R2

49.7 38

Mo MDC

11b

MDC

GFP CD11b

49.7

CD CD11c

MDC

CD11b CD11c

Class II CD86 Ly6C

slide-27
SLIDE 27

CD11b+CD11c+GFP+ cells transport CD11b 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 10 2 10 3 GFP 5 10 15 # Cells 2.28 0.5 1 1.5 # Cells 95.2 50 100 # Cells 48.2 200 10 2 10 3 10 4 10 5 CD11b 10 5 1.4 0 10 2 10 3 10 4 10 5 AF633-Conidia 20 25 10 3 10 4 10 5 CD11c 10 3 10 4 10 5 CD11c 50 100 150 # Cells 52.2 47.8 10 2 10 3 10 4 GFP 5 10 15 20 # Cells 0.018 10 3 10 4 10 5 CD11c 10 2 10 3 10 4 10 5 CD11b 0 10 2 10 3 10 4 10 5 Conidia 5

slide-28
SLIDE 28

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

slide-29
SLIDE 29

Ablation of Lung DC subsets in CCR2 Depleter

slide-30
SLIDE 30

CCR2 Depleter mice cannot prime A. f i t ifi CD4 T ll fumigatus-specific CD4 T cell responses

CCR2 depleter or C57BL/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 0.06 0.09 0.09 0.12 0.12 Cells Cells 10 103 10 104 10 105 hy 1. y 1.1 0.15 0.15

CCR2 Depleter (DT t t d) mLN CD4 Gate

DT DT

1 103 10 104 10 105 CFSE CFSE 0.03 0.03 # # 1 102 10 103 10 104 10 105 Thy 1.2 Thy 1.2 10 102 10 10 Th

(DT-treated)

6 9 12 12 Cells Cells 10 103 10 104 10 105 y 1. y 1.1 3.66 3.66

Non-Tg Control (DT t t d)

1 103 10 104 10 105 CFSE CFSE 3 6 # # 1 102 10 103 10 104 10 105 Thy 1.2 Thy 1.2 10 102 10 10 Th Th

(DT-treated)

slide-31
SLIDE 31

Summary

  • CCR2 reporter and depleter mice represent

valuable tools to dissect the role of monocytes and monocyte derived cells in microbial defense and monocyte-derived cells in microbial defense

  • Monocyte-derived lung DCs (CD11b+) transport

conidia to draining lymph nodes g y p

  • Ablation results in loss of A. fumigatus-specific

CD4 T cell priming M t i t ib t t i i d A

  • Monocytopenia may contribute to impaired A.

fumigatus CD4 T cell responses in patients undergoing HSCT g g

slide-32
SLIDE 32

Acknowledgements Acknowledgements

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