FIGURE 1.

Monocytes are necessary and sufficient for the detection of cffDNA through activation of the cGAS/STING pathway. (A) Maternal PBMCs collected at 36–38 wk gestation were incubated with 750 ng/ml cffDNA isolated from maternal plasma samples collected at 36–38 wk gestation (“3rd trimester”), 6 wk postpartum (“Postpartum”), or from term CTB supernatants (“CTB”). After overnight incubation, IL-1β was measured in PBMC supernatants by ELISA (n = 5–7 PBMC donors). Data are displayed as medians with IQRs. The p values were compared by Kruskal–Wallis test, with correction using false discovery rate for multiple comparison testing and two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. Significant comparisons (p ≤ 0.05) are indicated by third trimester versus postpartum (≠) and postpartum versus CTB (Δ). (B) Maternal PBMCs were stimulated overnight with 750 ng/ml cffDNA isolated from term CTBs. After overnight incubation with Effectene alone (“Media”) or Effectene with cffDNA (“cffDNA”), IL-1β, CXCL10, IL-18, and TNF-α levels were measured in PBMC supernatants (n = 4–5 PBMC donors). Data were presented as medians and IQRs, and p values were calculated using Mann–Whitney U test with ***p < 0.0005 and ****p < 0.0001. (C) PBMCs from a healthy female donor were sorted into populations of B cells (CD20⁺), NK cells (CD56⁺), monocytes (CD14⁺), and T cells (CD3⁺). Each cell type was stimulated with cffDNA overnight, and cytokine levels were quantified in duplicate using multiplex analysis. (D) Maternal PBMCs isolated from blood collected at 36–38 wk gestation were used to isolate monocytes (Mono) or monocyte-depleted PBMCs (PBMCs—Mono), then stimulated with cffDNA overnight. ELISA was used to measure IL-1β and CXCL10 concentrations in supernatants (n = 3–5 donors). Data are presented as medians and IQRs, and p values were calculated using Mann–Whitney tests: **p < 0.005, ****p < 0.0001. (E) Monocytes from three healthy female donors and one pregnant donor were incubated for 4 h with media (NT), Effectene reagent (E), or Effectene reagent with cffDNA DNA (E+cffDNA). After incubation, cells were harvested and lysed for Western blot analysis of IRF3, p-IRF3, and GAPDH expression (n = 4 donors). (F) Densitometry analysis was performed using Azure software and normalized to GAPDH expression. (G) Maternal monocytes were stimulated with either Effectene reagent alone or Effectene with cffDNA for 4 h. After incubation, cells (n = 3 donors, 4 replicates each) and supernatants (n = same 3 donors, 8 replicates each) were harvested to measure levels of active caspase-1. Media alone containing no cells was used as a blank. Data are represented as medians and IQRs, with p values calculated using Kruskal–Wallis tests. Significant comparisons (p ≤ 0.05) are indicated by Effectene (Media Blank) versus Monocytes+Effectene+cffDNA (≠) and Monocytes+Effectene versus Monocytes+Effectene+cffDNA (Δ).