Figure 4. Cathepsin B activity as well as ROS production are involved in αSyn F-induced inflammasome activation.

(A) Phagocytosis of fibrillar αSyn leads to phago-lysosomal destabilization. Flow cytometry of monocytes stained with acridine orange and then treated for 2 or 6 h with 40 nM αSyn F, 1 µM αSyn M. Data are reported as the percentage of acridine orange-positive cells present in the vehicle-exposed sample. Results are the mean ± S.D. of 3 experiments conducted in duplicate with cell preparations obtained from 3 different donors. ***p<0.001. (B) Monocytes were incubated for 30 min with 10 µM CA-074-Me (an inhibitor of cathepsin B), or left untreated; cells were then exposed for 6 h to 40 nM αSyn F or vehicle. IL-1β released into the culture supernatants was evaluated both by ELISA and immunoblot analysis. The same supernatants were assessed for the accumulation of active caspase-1 by immunoblot. Results are the mean ± S.D. of 3 experiments conducted in duplicate with cell preparations obtained from 3 different donors. The immunoblot is from one representative donor. ***p<0.001. (C) Monocytes were cultured for 1, 2 or 3 h in the presence of 1 µg/ml LPS or 40 nM αSyn F and intracellular ROS levels were quantified by the H₂DCF-DA fluorometric method. When required, monocytes were pre-incubated for 30 min with 20 µM DPI. The dotted line refers to the basal levels of ROS production (as in untreated monocytes). Results are the mean ± S.D. of 5 experiments conducted in duplicate with cell preparations obtained from 5 different donors. *p<0.05, **p<0.01 was calculated for samples not exposed to DPI vs the correspondent samples with DPI. (D) Monocytes were pre-incubated for 30 min with 20 µM DPI, or left untreated, before being exposed for 6 h to αSyn F or vehicle. IL-1β, as well as activated caspase-1, were assessed as before. Results are the mean ± S.D. of 3 experiments conducted in duplicate with cell preparations obtained from 3 different donors. The immunoblot is from one representative donor. **p<0.01.