FIGURE 3.

(a) 16 hr or (b) 24 hr exposure of primary mouse microglia to α‐syn fibrils and monomers (144.6 or 361.5 μg/ml nominal concentration). Fibrils induce IL‐1β secretion from the microglia whereas monomers do not. Points indicate mean of three replicates from independent microglia cultures generated from ≥6 mice each. (c) Confocal microscopic overview of NLRP3 (gray), ASC (red), and caspase‐1 (green) overlap illustrating inflammasome complex formation in primary mouse microglia treated for 16 hr with 144.6 μg/ml α‐syn fibrils. (d) Overnight exposure of PMA‐primed THP‐1 cells to a concentration gradient of α‐syn fibrils (fib., 72.3–289.2 μg/ml nominal concentration) induces dose‐dependent IL‐1β secretion. α‐Syn monomers (mon., 72.3–289.2 μg/ml) also induce some IL‐1β production from these cells at higher concentrations. **** = p <.0001; three experiments, n = 3. Colors indicate individual experiments. (e) Overnight exposure of primary human microglia to the same concentration gradient of α‐syn fibrils also induces dose‐dependent IL‐1β secretion. α‐Syn monomers, however, do not induce IL‐1β production at any concentration tested. ** = p <.01; 3 experiments, n = 3. Colors indicate individual experiments. (f) IL‐1β western blot of combined, concentrated supernatants from several primary human microglia experiments showing secretion of p35 pro‐IL‐1β and the mature p17 form upon noncanonical LPS‐mediated NLRP3 inflammasome activation (20 ng/ml overnight) or α‐syn‐mediated NLRP3 inflammasome activation (72.3 μg/ml overnight). (g–i) quantification of p35 (g), p26 (h), and p17 (i) IL‐1β bands from western blot (f) after overnight treatment of primary human microglia with 20 ng/ml LPS or 72.3 μg/ml α‐syn [Color figure can be viewed at wileyonlinelibrary.com]