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. 2021 Nov 15;207(10):2433–2444. doi: 10.4049/jimmunol.2100649

FIGURE 4.

FIGURE 4.

The inflammatory response to cffDNA induces myometrial contraction in vitro. (A) Maternal PBMCs from 36 to 38 wk gestation were stimulated overnight with Effectene reagent alone or Effectene reagent with cffDNA (n = 6). Supernatants (CM or cffDNA CM) were then added to PHM1-41 cells embedded in a collagen matrix, and contraction was measured after overnight incubation as the distance between the edge of the well and the collagen–cell matrix. Representative wells are shown. (B) Third trimester maternal PBMCs were stimulated overnight with Effectene reagent alone or Effectene reagent with cffDNA (n = 4 donors) as in (A). Resulting supernatants (CM or cffDNA CM) were then added to PHM1-41 cells embedded in a collagen matrix with or without TNF-α neutralizing Abs for 18 h, and contraction was measured as the distance between the edge of the well and the collagen–cell matrix. Representative wells are shown. (C) Maternal PBMCs (n = 3 donors) were stimulated with Effectene or Effectene+cffDNA overnight. PBMC supernatants (CM and cffDNA CM) were harvested for coculture with PHM1-41 cells in a 24-well plate. Levels of PGE2 were measured in PHM1-41 supernatants after overnight incubation with cffDNA CM supernatants with or without TNF-α blocking Ab. The p values were calculated using Kruskal–Wallis test. Significant comparisons (p ≤ 0.05) are indicated by CM versus cffDNA CM (≠) and cffDNA CM versus TNF-α block (Δ). (D) Maternal monocytes from blood collected at 36–38 wk gestation were stimulated overnight with Effectene reagent alone or Effectene reagent with cffDNA (n = 6 donors). Resulting monocyte supernatants (CM or cffDNA CM) were added to PHM1-41 cells embedded in a collagen matrix. Contraction was measured after overnight coculture of CM with the PHM1-41 collagen matrix. (E) Maternal monocytes from 36–38 wk gestation were stimulated overnight with Effectene reagent alone or Effectene reagent with cffDNA (n = 4 donors). CM or cffDNA CM were then added to PHM1-41 cells embedded in a collagen matrix with or without neutralizing Abs for IL-1β. For all data in this figure, the matrices were imaged and contraction was measured after overnight (18 h) incubation as the distance between the edge of the well and the collagen–cell matrix. The p values were calculated using Mann–Whitney tests, ****p < 0.0001; graphs display median and IQR. (F) Maternal monocytes (n = 3 donors) were stimulated with Effectene or Effectene and cffDNA overnight. Monocyte supernatants (CM or cffDNA CM) were harvested for overnight coculture with PHM1-41 cells with or without IL-1β blocking Abs in a 24-well plate. Levels of PGE2 were measured by ELISA. The p values were calculated using Kruskal–Wallis test. Significant comparisons (p ≤ 0.05) are indicated by CM versus cffDNA CM (≠) and cffDNA CM versus IL-1β block (Δ).