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

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

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

Live Conidia induce Airway Neutrophil Live Conidia induce Airway Neutrophil Recruitment 42 Total Cells intratracheal Macrophages 36 Neutrophils Neutrophils 30 24 18 12 6 Vehicle Heat-killed Live Conidia Resting conidia Hohl, T.M. et al., PloS Pathog. 1:e30, 2005.

Live Conidia induce TNF/ CXCL2 Secretion Live Conidia induce TNF/ CXCL2 Secretion by Alveolar Macrophages TNF CXCL2 60 24 45 18 ng/ml 30 12 15 6 Live HK Medium LPS Live HK Medium LPS Hohl, T.M. et al., PloS Pathog. 1:e30, 2005.

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

Killed swollen Conidia induce Neutrophil Killed swollen Conidia induce Neutrophil Influx into the BAL fluid 30 Total Cells Macrophages Neutrophils 24 18 12 6 Live Heat-killed Heat killed Conidia swollen conidia Hohl, T.M. et al., PloS Pathog. 1:e30, 2005.

Swollen Conidia and Germlings Swollen Conidia and Germlings expose β -glucan on their surface Anti β -glucan Anti β -glucan Isotype Control Ab Isotype Control Ab Hohl, T.M. et al., PloS Pathog. 1:e30, 2005.

Dectin-1 binds and signals in response to β -(1,3) glucan β (1 3) i t l Brown, G. D., Nat Rev Immunol 6:33-43, 2006.

Conidia Stimulate Dectin 1 and MyD88 Conidia Stimulate Dectin-1- and MyD88- dependent Pathways TNF CXCL2 3 3 2 2 /ml ng/ 1 1 anti-Dectin - + - + - + - + MyD88 -/- MyD88 -/- WT WT Hohl, T.M. et al., PloS Pathog. 1:e30, 2005.

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

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 Echinocandins do not fully inhibit A. fumigatus growth, yet induce di d t f ll i hibit A f i t th t i d prominent morphologic changes at or above the MEC 1 x MEC Caspofungin (63 ng/ml) No Caspofungin

Caspofungin Decreases Macrophage Inflammatory Responses to Conidia Responses to Conidia 1.8 ng/ml) 1.2 No Caspofungin No Caspofungin TNF (n * * * * 0.6 Caspo (ng/ml) 0 4 8 16 31 63 125250500 Caspofungin (500 ng/ml) BMM φ TNF/CXCL2 release (500 ng/ml caspofungin vs. no drug exposure): 0.49 ± 0.04* (range 0.46-0.54; n=4) • TNF 0.55 ± 0.10* (range 0.43-0.62; n=4) • CXCL2 Hohl, T.M. et al. J Infect Dis , 2008.

Caspofungin Enhances Macrophage Inflammatory Caspofungin Enhances Macrophage Inflammatory Responses to Hyphae 10 8 * * * * * * * * g/ml) 8 ml) 6 TNF (ng/m CXCL2 (ng 6 4 4 2 2 C Caspo (ng/ml) 0 4 8 16 31 63 125 250 500 0 4 8 16 31 63 125 250 500 BMM φ TNF/CXCL2 release (500 ng/ml caspofungin vs. no drug exposure) 4.11 ± 2.39* (range 1.90-7.84; n=8) • TNF 2.90 ± 1.40* (range 1.53-5.41; n=8) • CXCL2 Hohl, T.M. et al. J Infect Dis , 2008.

Caspofungin Modulates Dectin-1-dependent Inflammatory Responses to Conidia, Germlings, and Hyphae Responses to Conidia Germlings and Hyphae Conidia Germlings Hyphae 1.2 3.5 7 3 3 6 6 NF (ng/ml) 2.5 5 0.8 2 4 1.5 3 TN 0.4 0 4 1 1 2 2 0.5 1 Caspofungin - + - + - + 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 Exposure Effects of Echinocandin Drugs on β -glucan Exposure Caspofungin No Caspofungin anti- β -glucan anti- β -glucan DIC DIC A 8 h 10 h

Effects of Echinocandin Drugs on β -glucan Exposure Caspofungin Caspofungin No Caspofungin No Caspofungin anti- β -glucan anti- β -glucan DIC DIC A 12 h 15 h 18 h Hohl, T.M. et al. J Infect Dis , 2008.

Quantitative Analysis of β -glucan Immunoreactivity associated f β Q tit ti A l i l I ti it i t d with Caspofungin-treated and Untreated Hyphae Integrated Fluorescence Intensity/Fungal Mass (Arbitrary Units) Caspofungin-treated Untreated Hyphae Hyphae 21.4 ± 8.3* 1.83 ± 0.73 Expt. 1 43.7 ± 7.0* 2.96 ± 4.67 Expt. 2 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 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

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

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

Recruitment of GFP + monocytes and myeloid DCs into the lungs of A. fumigatus- infected mice g g A. fumigatus 14.6 31.4 MDCs (2.7 M) Mo (5.2 M) 60.6 Uninfected 29.1 7.2 MDCs(0.4 M) D11c GFP CD G Mo (0 7 M) Mo (0.7 M) 48 48 Ly6G CD11b

Cell Recruitment to mLN after intratracheal i f infection with A. fumigatus conidia ti ith A f i t idi Uninfected A. fumigatus 0.52 M 2.24 M 4.16 M 0.25 M 5 5 5 5 5 5 5 5 1.09 1 09 1 17 1.17 0.21 0 21 0 04 0.04 10 10 10 10 GFP 4 4 4 4 10 10 10 10 3 3 3 3 10 10 10 10 2 2 2 2 10 10 10 10 0 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 CD11b 50 GFP + cells Infected 0 3 cells) 40 Uninfected 30 (x 10 CD11b + 20 10

Characterization of GFP + cells in the mLN 48 h 48 h post-infection with A. fumigatus t i f ti ith A f i t Mo 38 1.17 Mo R2 MDC 1.17 11b GFP P 49.7 49.7 CD MDC MDC CD11b CD11b CD11c CD11c Class II CD86 Ly6C

CD11b + CD11c + GFP + cells transport CD11b CD11c GFP cells transport labelled conidia to mLN 25 10 5 150 2.5 0.4 20 10 4 2 51.8 100 100 15 15 P GFP # Cells # Cells # Cells 1.5 2.28 10 3 48.2 95.2 10 1 50 10 2 5 0.5 0 0 0 0 0 0 0 10 2 10 3 10 4 10 5 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 10 3 10 4 10 5 0 0 0 CD11b CD11c AF633-Conidia CD11c 25 10 5 200 1.4 20 20 10 4 150 47.8 GFP 15 # Cells # Cells 0.018 10 3 52.2 100 10 10 2 50 5 5 0 0 0 10 2 10 3 10 4 10 5 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 0 CD11b CD11c Conidia

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

Ablation of Lung DC subsets in CCR2 Depleter

CCR2 Depleter mice cannot prime A. fumigatus -specific CD4 T cell responses f i t ifi CD4 T ll AF-specific CD4 T cells (Thy 1.1/1.2) CCR2 depleter or C57BL/6 (Thy 1.2) Infection via i.t. route -1 0 +1 +6 mLN CD4 Gate mLN CD4 Gate DT DT 0.12 0.12 0.15 0.15 10 5 10 0.09 0.09 10 4 10 CCR2 Depleter y 1.1 Cells Cells hy 1. 10 3 0.06 0.06 10 10 10 (DT t (DT-treated) t d) Th # # 10 2 0.03 0.03 10 0 0 10 2 10 3 10 4 10 5 10 3 10 4 10 5 0 0 1 10 10 10 0 0 1 10 10 Thy 1.2 Thy 1.2 CFSE CFSE 3.66 3.66 10 5 12 12 10 10 4 9 10 Non-Tg Control y 1.1 Cells Cells y 1. 10 3 6 6 10 10 10 (DT-treated) (DT t t d) Th Th # # 10 2 10 3 0 0 10 2 10 3 10 4 10 5 10 3 10 4 10 5 0 0 1 10 10 10 0 0 1 10 10 Thy 1.2 Thy 1.2 CFSE CFSE

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 • Monocytopenia may contribute to impaired A. M t i t ib t t i i d A fumigatus CD4 T cell responses in patients undergoing HSCT g g