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. 2013 Aug 1;191(3):1287-99.
doi: 10.4049/jimmunol.1300132. Epub 2013 Jul 1.

Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling

Irene Kyrmizi  1 , Mark S Gresnigt, Tonia Akoumianaki, George Samonis, Prodromos Sidiropoulos, Dimitrios Boumpas, Mihai G Netea, Frank L van de Veerdonk, Dimitrios P Kontoyiannis, Georgios Chamilos
Affiliations

Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling

Irene Kyrmizi et al. J Immunol. .

Abstract

Aspergillus fumigatus is the predominant airborne fungal pathogen in immunocompromised patients. Genetic defects in NADPH oxidase (chronic granulomatous disease [CGD]) and corticosteroid-induced immunosupression lead to impaired killing of A. fumigatus and unique susceptibility to invasive aspergillosis via incompletely characterized mechanisms. Recent studies link TLR activation with phagosome maturation via the engagement of autophagy proteins. In this study, we found that infection of human monocytes with A. fumigatus spores triggered selective recruitment of the autophagy protein LC3 II in phagosomes upon fungal cell wall swelling. This response was induced by surface exposure of immunostimulatory β-glucans and was mediated by activation of the Dectin-1 receptor. LC3 II recruitment in A. fumigatus phagosomes required spleen tyrosine kinase (Syk) kinase-dependent production of reactive oxygen species and was nearly absent in monocytes of patients with CGD. This pathway was important for control of intracellular fungal growth, as silencing of Atg5 resulted in impaired phagosome maturation and killing of A. fumigatus. In vivo and ex vivo administration of corticosteroids blocked LC3 II recruitment in A. fumigatus phagosomes via rapid inhibition of phosphorylation of Src and Syk kinases and downstream production of reactive oxygen species. Our studies link Dectin-1/Syk kinase signaling with autophagy-dependent maturation of A. fumigatus phagosomes and uncover a potential mechanism for development of invasive aspergillosis in the setting of CGD and corticosteroid-induced immunosupression.

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Figures

Figure 1
Figure 1. LC3 II is selectively recruited to phagosomes of primary human monocytes during cell wall swelling ofA. fumigatus
A-B. Primary human monocytes (2 × 105 cells/condition) isolated from healthy individuals were infected with live GFP A. fumigatus (GFP Af; A, B) or PFA-killed GFP A. fumigatus (B) at a MOI 5: 1 for the indicated times. Cells were fixed, permeabilized, stained for LC3 II with the use of an Alexa555 secondary antibody (red) and TOPRO-3 (blue, nuclear staining) and analyzed by immunofluorescence confocal microscopy. The percentages of LC3-associated A. fumigatus-containing phagosomes (LC3+Aspergillus; n > 150 per group) at all time points were quantified by measuring the number of LC3+Aspergillus-containing phagosomes out of the total number of engulfed Aspergillus spores and data are presented as mean + S.E.M. of 3 independent experiments. *, P < 0.0001, paired Student’s t test. Bar, 5 µm. C. Primary human monocytes (2 × 106 cells/condition) were infected with live GFP A. fumigatus or PFA-killed GFP A. fumigatus as in A-B for the indicated times. Cell lysates were prepared and levels of LC3 II protein were determined by immunoblotting. Levels of tubulin in the same lysates were determined by immunoblotting as loading controls. D-E. Primary human monocytes were stimulated for 1h with PFA-killed dormant or PFA-killed swollen spores of GFP A. fumigatus, fixed and stained as in A. The percentages of LC3+A. fumigatus-containing phagosomes (LC3+Aspergillus; n > 150 per group) were quantified and data are presented as mean + S.E.M. of 5 independent experiments. *, P < 0.0001, paired Student’s t test. Bar, 5 µm. F. Primary human monocytes (2 × 106 cells/condition) were left untreated (unstim) or stimulated with either PFA-killed dormant or PFA-killed swollen spores of A. fumigatus. Cell lysates were prepared and LC3 II and tubulin protein levels were determined by immunoblotting. G. Representative immunoelectron micrograph in which LC3 II was labeled in primary human monocytes stimulated for 1h with PFA-killed A. fumigatus swollen spores with 1.4-nm gold particles.
Figure 2
Figure 2. β-glucan surface exposure in swollen spores of A. fumigatus triggers LC3 II recruitment in fungal phagosomes
A. Primary human monocytes (2 × 105 cells/condition) isolated from healthy individuals were infected with GFP A fumigatus swollen spores with or without laminarin (500 µg/ml) or swollen spores following overnight enzymatic digestion of β-glucan (β-glucanase) at a MOI 5: 1 for 1h. Cells were fixed, permeabilized, stained for LC3 II with the use of an Alexa555 secondary antibody (red) and TOPRO-3 (blue, nuclear staining) and analyzed by immunofluorescence confocal microscopy. Bar, 5 µm. B The percentages of LC3+A. fumigatus-containing phagosomes (LC3+Aspergillus n > 150 per group) were quantified and data are presented as mean + S.E.M. of 3 independent experiments. *, P < 0.0001, paired Student’s t test. C Primary human monocytes (2 × 106 cells/condition) were stimulated with A. fumigatus swollen spores alone or in the presence of increasing concentrations of laminarin, or (D) IgG coated 3mm latex beads alone or in the presence of increasing concentrations of laminarin for 1h. Cell lysates were prepared and levels of LC3 II protein were determined by immunoblotting. Levels of tubulin in the same lysates were determined by immunoblotting as loading controls. E-F. Primary human monocytes (2 × 105 cells/condition) were left untreated or stimulated with purified β-gucan (curdlan, 100 µg/ml) or LPS (100 ng/ml) with or without pretreatment with laminarin (500 µg/ml). The percentages of human monocytes containing autophagosomes as indicated by punctuate LC3 staining (LC3+ monocytes; n > 150 per group) were quantified and data are presented as mean + S.E.M. of 2 independent experiments. *, P < 0.0001, paired Student’s t tes. Bar, 5 µm (G). Primary human monocytes (2 × 105 cells/condition) were stimulated with FITC-labeled BSA beads or DTFA-labeled WGP at a MOI 5: 1 for 1h. Cells were processed as in A and analyzed by immunofluorescence confocal microscopy. Bar, 5 µm. (H). Primary human monocytes (1 × 106 cells/condition) were left untreated or stimulated with BSA coated-beads, IgG coated-beads or WGP with or without pretreatment with increasing concentration of laminarin at a MOI of 10:1 for 1h. Cell lysates were prepared and levels of LC3 II and tubulin were determined by immunoblotting.
Figure 3
Figure 3. Dectin-1/syk kinase signalling regulates LC3 II recruitment in A. fumigatus phagosomes
A. Primary human monocytes (2 × 105 cells/condition) isolated from homozygous patients with the stop codon mutation 238X (Dectin-1 −/−) and healthy controls (Dectin-1 +/+) were infected with FITC-labeled resting or swollen spores of A. fumigatus at a MOI 5: 1 for 1h at 37 °C. Cells were fixed, permeabilized, and stained for LC3 II as in Figure 1A. The percentages of LC3+A. fumigatus-containing phagosomes (LC3+Aspergillus; n > 100 per group) were quantified and data are presented as mean + S.D. for each patient. *, P < 0.0001, paired Student’s t test. B. Primary human monocytes from healthy individuals were stimulated with FITC-labeled swollen spores of A. fumigatus following 30 min pre-incubation with blocking antibodies for Dectin-1 (10 µg/ml), TLR-2 (10 µg/ml), or TLR-4 (10 µg/ml) or the indicated isotype control antibodies (10 µg/ml) at a MOI 5: 1 for 1h at 37 °C. Cells were processed for immunofluorescence microscopy as in Fig 1A. C. Primary human monocytes (2 × 106 cells/condition) from healthy individuals were either left untreated, or stimulated with resting spores of A. fumigatus, or swollen spores of A. fumigatus with or without 30 min pretreatment with syk inhibitor (1µM) at a MOI 10:1 for 10 min at 37 °C. Cell lysates were prepared and levels of phospho-syk activity were determined by immunoblotting. Levels of tubulin and total syk in the same lysates were determined by immunoblotting as loading controls. D. Primary human monocytes (2 × 106 cells/condition) were stimulated as in C for 1h at 37 °C and levels of LC3 II and tubulin were determined in cellular lysates by immunoblotting. E-F. Primary human monocytes (2 × 105 cells/condition) were stimulated with FITC-labeled swollen spores of A. fumigatus with or without 30 min pretreatment with syk inhibitor (1µM) or raf-1 inhibitor (40µM) at a MOI of 5:1 for 1h and processed for immunostaining as in A. The percentages of LC3+A. fumigatus-containing phagosomes (LC3+Aspergillus; n > 150 per group) were quantified and data are presented as mean + S.E.M. of four independent experiments. *, P < 0.0001, paired Student’s t test. Bar, 5 µm. G. Primary human monocytes (2 × 106 cells/condition) stimulated as in E and levels of LC3 II and tubulin were determined in cellular lysates by immunoblotting.
Figure 4
Figure 4. Syk kinase dependent ROS production regulates formation of LC3+ Aspergillus containing phagosomes
A. Primary human monocytes (2 × 105 cells/condition) were left unstimulated or infected with resting spores or swollen spores of A. fumigatus at a MOI of 5:1 with or without 30 min pretreatment with syk inhibitor (1 µM) or stimulated with PMA (100 ng/ml) with or without 30 min pretreatment with syk inhibitor (1 µM) for 1h at 37 °C. DCFH-DA was added during the last 30 min of stimulation and intracellular ROS production was determined by measurement of relative fluorescent intensity at the FL1 channel (log MFI). Differences in ROS production between experimental groups were quantified and data are presented as mean + S.E.M. from 4 independent experiments. *, P < 0.005, paired Student’s t test. Representative FL1 histograms from human monocytes left untreated (gray solid line), stimulated with either swollen spores of A. fumigatus alone or PMA alone (black solid line) or in the presence of syk inhibitor (black dashed lines) are shown. B-C. Primary human monocytes (2 × 105 cells/condition) isolated from CGD patients and healthy controls were infected with FITC-labeled resting or swollen spores of A. fumigatus at a MOI 5: 1 for 1h at 37 °C. Cells were fixed, permeabilized, and stained for LC3 II as in Figure 1A. The percentages of LC3+A. fumigatus-containing phagosomes (LC3+Aspergillus; n > 100 per group) were quantified and data are presented as mean + S.D. for each patient. *, P < 0.0001, paired Student’s t test. Representative immunofluorescence image of LC3+ phagosomes containing FITC-labeled swollen spores of A. fumigatus in monocytes obtained from healthy control and CGD patient. D. Primary human monocytes (1 × 106 cells/condition) from a representative CGD patient and the corresponding healthy control were left untreated (unsitm) or stimulated with resting spores or swollen spores of A. fumigatus at a MOI 10:1 for 1h at 37 °C and levels of LC3 II and tubulin were determined in cellular lysates by immunoblotting.
Figure 5
Figure 5. Conditional inactivation of Atg5 in THP-1 human macrophages results in attenuated phagolysosomal fusion and killing of A. fumigatus
A. THP-1 cells (1 × 106 cells/condition) were transfected with RNAi sequences targeting ATG5 vs. scramble control RNAi (C RNAi) by Amaxa electroporation. Cell lysates were prepared 48 h following transfection and levels of LC3 II, Atg5, and Atg5-Atg12 proteins were determined by immunoblotting. Levels of actin in the same lysates were determined by immunoblotting as loading controls. B-C. LysoTracker staining in THP-1 cells transfected with ATG5 RNAi or C RNAi and differentiated to macrophages with addition of PMA (25 ng/ml) following 2h of infection with FITC-labeled A. fumigatus spores. Data are presented as mean + S.E.M. of three independent experiments. *, P < 0.0001, paired Student’s t test. Bar, 5 µm. D. Degree of association (uptake) of GFP A. fumigatus spores with THP-1 cells transfected with ATG5 RNAi or C RNAi and differentiated to macrophages in the presence of PMA (25 ng/ml) at different time points of infection (1h, 2h, 6h), assessed by FACS analysis. Results are representative of two independent experiments. E. THP-1 cells transfected by Amaxa nucelofection with ATG5 RNAi or C RNAi, were seeded in 12 well plates (5 × 105 cells/condition), differentiated with PMA (25 ng/ml) for 48 h and infected with A. fumigatus spores at a MOI of 1:10 at 37 °C. Medium containing nonadherent, nonphagocytosed conidia was removed at 1 h, and wells were washed three times using warm PBS. Macrophages were then allowed to kill conidia for 2 h and 6 h before intracellular conidia were harvested. The percentage of germinating spores in the culture well after 6 to 8 h of incubation at 37°C was assessed under a microscope. The percentage of germination rate (number of germinated spores per 100 counted conidia) of A. fumigatus spores following different time points of infection (1 h, 2 h, 6 h) was calculated and data are expressed as mean + S.E.M. of three independent experiments; * P = 0. 0003, paired Student’s t test.
Figure 6
Figure 6. Corticosteroids block LC3 II recruitment in A. fumigatus phagosomes via inhibiting phosphorylation of syk kinase and downstream ROS production
A. Primary human monocytes (2 × 105 cells/condition) from six consecutive patients with rheumatologic diseases were collected before and 2h after intravenous administration of corticosteroids (250 mg hydrocortisone) and stimulated with swollen spores of A. fumigatus at a MOI 5:1 at 37 °C. Cells were fixed, permeabilized, stained for LC3 II with the use of an Alexa555 secondary antibody (red) and TOPRO-3 (blue, nuclear staining) and analyzed by immunofluorescence confocal microscopy. The percentages of LC3+A. fumigatus-containing phagosomes (LC3+Aspergillus; n > 150 per group), before (0h) and after (2h) corticosteroid treatment, were quantified and data are presented as mean + S.D. for each patient. *, P < 0.05, paired Student’s t test. B. Representative immunofluorescence image of LC3+ phagosomes containing FITC-labeled swollen spores of A. fumigatus in monocytes obtained before (0h) and after (2h) administration of corticosteroids. Bar, 5 µm. C. Primary human monocytes (2 × 105 cells/condition) from healthy individuals (n = 4) were stimulated before (0h) and after (2h) ex vivo exposure to hydrocortisone (20 µg/ml), fixed and processed as in A; data are presented as mean + S.E.M. of four independent experiments. P < 0.05, paired Student’s t test. D. Primary human monocytes (2 × 106 cells/condition) from healthy individuals were either left untreated with or without 1h exposure to hydrocortisone (20 µg/ml), or stimulated with swollen spores of A. fumigatus with or without 1h pre-exposure to hydrocortisone ((20 µg/ml) at a MOI 10:1 for 10 min at 37 °C. Cell lysates were prepared and levels of phospho-syk activity were determined by immunoblotting. Levels of tubulin and total syk in the same lysates were determined by immunoblotting as loading controls. E. Primary human monocytes (2 × 105 cells/condition) were left unstimulated or infected with swollen spores of A. fumigatus at a MOI of 5:1 for 1 h with or without pre-exposure (2 h) to increasing concentrations of hydrocortisone at 37 °C. DCFH-DA was added during the last 30 min of stimulation and intracellular ROS production was determined by measurement of relative fluorescent intensity at the FL1 channel (log MFI). Representative FL1 histograms from human monocytes left untreated (gray area), or stimulated with swollen spores of A. fumigatus (black solid line) with or without pre-exposure to hydrocortisone (20 µg/ml) are shown. Differences in ROS production between experimental groups were quantified and data are presented as mean + S.E.M. from 4 independent experiments. *, P < 0.0001, paired Student’s t test.
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