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.