Figure 1.
Inflammasome activation induces loss of placental TM and trophoblast proliferation. (A) Stacked bar graph showing the frequency of embryos (red, TM+/+; green, TM−/−; blue, TM+/−). Anakinra treatment (either 20 or 40 mg/kg body weight, once daily starting on day 7.5 postcoitus) or genetic NLRP3 deficiency (Nlrp3−/−) do not rescue TM-null embryos (green bar) from lethality (n = 5 mothers per group). (B-C) Immunoblotting analysis (B, representative immunoblots; C, bar graph summarizing the results) showing a dose- and time-dependent (24 and 48 hours) reduction in TM expression upon treatment of differentiated mTS cells with exogenous IL-1β (n = 3 independent experiments). *P < .05 (relative to control; C, 24 hours); #P < .05 (relative to control; C, 48 hours), untreated controls, ANOVA (C). (D-G) Endothelial cell–derived EVs reduce TM expression in mice (D-E, n = 8 placentae from 3 different mothers), and anakinra treatment of EV-injected pregnant C57BL/6 mice restores TM expression. EVs reduce TM expression in differentiated mouse trophoblast cells (F-G, mTS; n = 3 independent experiments) or in human trophoblast-like cells (F-G, JEG-3; n = 3 independent experiments). Representative immunoblots (D,F). Bar graph summarizing the results (E). Line graph summarizing the results (G). *P < .05; Student t test (E); ANOVA (G). (H-J) Analysis of human placenta samples (n = 14 per group). Reduced TM expression (H) in PE patients compared with healthy pregnant controls (C), inverse correlation of TM expression and IL-β levels (I, PE patients, lavender; healthy pregnant controls [C], green), and positive correlation of TM expression and platelet counts (J). **P < .01; Student t test (H); Pearson correlation (I-J). (K-L) Ki-67 staining (brown, Ki-67+ nuclei; blue, hematoxylin nuclear counterstaining) in human placenta (K, representative images taken at ×40 magnification; L, dot plot with Pearson correlation; n = 14 per group) showing a positive association of TM expression and cell proliferation (PE patients [lavender]; healthy pregnant controls, C [green]; arrows, Ki-67+ nuclei). (M-O) Treatment of EV-injected pregnant C57BL/6 mice (M-N, n = 8 placentae from 3 different mothers per group) or differentiated mTS cells (O) with anakinra restores trophoblast cell proliferation (M, Ki-67 immunostaining, representative images taken at ×40 magnification; N, bar graph summarizing the results) and bromodeoxyuridine incorporation in mTS cells (O, bar graph summarizing the results). *P < .05 (relative to control [C]), #P < .05 (relative to EV); ANOVA (N,O). ANA, Anakinra; AU, arbitrary unit; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, nonsignificant, χ2 test; UT, untreated control.

Inflammasome activation induces loss of placental TM and trophoblast proliferation. (A) Stacked bar graph showing the frequency of embryos (red, TM+/+; green, TM−/−; blue, TM+/−). Anakinra treatment (either 20 or 40 mg/kg body weight, once daily starting on day 7.5 postcoitus) or genetic NLRP3 deficiency (Nlrp3−/−) do not rescue TM-null embryos (green bar) from lethality (n = 5 mothers per group). (B-C) Immunoblotting analysis (B, representative immunoblots; C, bar graph summarizing the results) showing a dose- and time-dependent (24 and 48 hours) reduction in TM expression upon treatment of differentiated mTS cells with exogenous IL-1β (n = 3 independent experiments). *P < .05 (relative to control; C, 24 hours); #P < .05 (relative to control; C, 48 hours), untreated controls, ANOVA (C). (D-G) Endothelial cell–derived EVs reduce TM expression in mice (D-E, n = 8 placentae from 3 different mothers), and anakinra treatment of EV-injected pregnant C57BL/6 mice restores TM expression. EVs reduce TM expression in differentiated mouse trophoblast cells (F-G, mTS; n = 3 independent experiments) or in human trophoblast-like cells (F-G, JEG-3; n = 3 independent experiments). Representative immunoblots (D,F). Bar graph summarizing the results (E). Line graph summarizing the results (G). *P < .05; Student t test (E); ANOVA (G). (H-J) Analysis of human placenta samples (n = 14 per group). Reduced TM expression (H) in PE patients compared with healthy pregnant controls (C), inverse correlation of TM expression and IL-β levels (I, PE patients, lavender; healthy pregnant controls [C], green), and positive correlation of TM expression and platelet counts (J). **P < .01; Student t test (H); Pearson correlation (I-J). (K-L) Ki-67 staining (brown, Ki-67+ nuclei; blue, hematoxylin nuclear counterstaining) in human placenta (K, representative images taken at ×40 magnification; L, dot plot with Pearson correlation; n = 14 per group) showing a positive association of TM expression and cell proliferation (PE patients [lavender]; healthy pregnant controls, C [green]; arrows, Ki-67+ nuclei). (M-O) Treatment of EV-injected pregnant C57BL/6 mice (M-N, n = 8 placentae from 3 different mothers per group) or differentiated mTS cells (O) with anakinra restores trophoblast cell proliferation (M, Ki-67 immunostaining, representative images taken at ×40 magnification; N, bar graph summarizing the results) and bromodeoxyuridine incorporation in mTS cells (O, bar graph summarizing the results). *P < .05 (relative to control [C]), #P < .05 (relative to EV); ANOVA (N,O). ANA, Anakinra; AU, arbitrary unit; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, nonsignificant, χ2 test; UT, untreated control.

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