Dectin-1 Pathway Activates Robust Autophagy-Dependent Unconventional Protein Secretion in Human Macrophages

Abstract

Dectin-1 is a membrane-bound pattern recognition receptor for β-glucans, which are the main constituents of fungal cell walls. Detection of β-glucans by dectin-1 triggers an effective innate immune response. In this study, we have used a systems biology approach to provide the first comprehensive characterization of the secretome and associated intracellular signaling pathways involved in activation of dectin-1/Syk in human macrophages. Transcriptome and secretome analysis revealed that the dectin-1 pathway induced significant gene expression changes and robust protein secretion in macrophages. The enhanced protein secretion correlated only partly with increased gene expression. Bioinformatics combined with functional studies revealed that the dectin-1/Syk pathway activates both conventional and unconventional, vesicle-mediated, protein secretion. The unconventional protein secretion triggered by the dectin-1 pathway is dependent on inflammasome activity and an active autophagic process. In conclusion, our results reveal that unconventional protein secretion has an important role in the innate immune response against fungal infections.

Global Characterization of Protein Secretion from Human Macrophages Following Non-canonical Caspase-4/5 Inflammasome Activation

“…1C). We have previously shown that TLR4 activation by extracellular LPS does not induce EV-mediated protein secretion in human macrophages (16). In contrast to this, the current results strongly suggest that intracellular LPS is a potent activator of EV-mediated protein secretion.…”

“…For this, macrophages were mock-transfected or transfected with LPS for different time periods, and the cell culture supernatants were collected and concentrated. The total secretome was separated into two fractions as follows: enriched extracellular vesicle (EV) fraction and rest-secretome (RS) fraction using sizeexclusion centrifugation as described previously (16). After this, the proteins in EV and RS fractions were separated by SDS-PAGE and visualized with silver staining.…”

“…Unconventionally secreted proteins lack the signal peptide and are secreted directly through the plasma membrane through vesicles, including exosomes derived from multivesicular bodies (13,14). Recent system-level studies of protein secretion by activated immune cells, including macrophages, have highlighted the importance of different secretory pathways in innate immune response (15)(16)(17)(18). We have shown that NLRP3 inflammasome activators ATP and crystallized monosodium urate induce robust protein secretion in human macrophages (19,20), but the effect of non-canonical inflammasome activation on global protein secretion has remained uncharacterized.…”

Mechanisms of unconventional secretion of IL-1 family cytokines

“…A major issue with this model is that the authors were unable to localise IL-1b to multivesicular bodies or exosomes, either before or after release. Another study was similarly unable to detect mature IL-1b/18 in exosomes purified from stimulated human macrophages [20]. Further, a later study by the Dubyak group showed that ATP-dependent secretion of MHC-IIcontaining exosomes does not require caspase-1 [21], but numerous reports have documented the requirement for caspase-1 for IL-1b secretion [7,15,22].…”

Immunologic manifestations of autophagy

“…Processed caspase-1 (p20) is reduced in cell extracts upon stimulation of autophagy (27). As described above, once the inflammasome is activated, autophagy contributes to the unconventional secretion of IL-1β as well as IL-18 and HMGB1 (17,18). An alternative can come directly from intracellular bacteria (38,39).…”