EVs cause inflammasome activation in placenta. (A-B) Inflammasome activation in murine placentae after EV injections (mouse endothelial cell–derived EVs). Immunoblots showing increased cleaved Casp-1 and cleaved IL-1β and increased NLRP3 expression in murine placentae after EV injections, analyzed at day 12.5 p.c. (A, representative immunoblots; B, bar graph summarizing results). Arrowheads indicate inactive (proform, white arrowheads) and the active (cleaved form, black arrowheads) form of Casp-1 or IL-1β, respectively (A). Only the active form was quantified (B). (C-D) Representative images of (top) uterus, (middle) placentae, and (bottom) embryos showing protection from mouse endothelial-derived EV-induced pregnancy complications in NLRP3^−/− (C) and Casp-1^−/− (D, Casp-1) mice. Size bar, 1 mm. (E) Double immunofluorescence staining showing activated platelets (CD62P, P-selectin; red) in the placenta after EV injection in pregnant NLRP3^−/− females mated with NLRP3^−/− males. Activated platelets are in direct contact with syncytiotrophoblast (Gcm-1; green); yellow indicates colocalization of activated platelets and Gcm-1⁺ syncytiotrophoblast (arrows); conventional immunofluorescence analyses; arrowheads, autofluorescence of erythrocytes. Scale bar, 20 µm. Control mice (C) were injected with the supernatant obtained after the last PBS wash during EV isolation. Data shown represent mean ± SEM of at least 8 placentae or embryos analyzed from at least 3 different litters of each group *P < .05 (relative to control, C). (B) Student t test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.