(706) Cell-free fetal DNA impairs trophoblast migration in a TLR9-dependent manner and is reversed by hydroxychloroquine

Friday, February 10, 2023

10:30 AM – 12:00 PM

Objective: Growing evidence suggests a relationship between elevated circulating cell-free fetal DNA (cffDNA) and preeclampsia. Hypomethylation of CpG motifs is a hallmark of cffDNA, allowing it to activate Toll-like receptor 9 (TLR9). Since TLR9 is expressed by the trophoblast, we hypothesize that placental-derived cffDNA accumulates at the maternal-fetal interface and limits trophoblast migration. We investigated this and if available therapeutics would have an impact.

Study Design: A human first trimester extravillous trophoblast cell line (Sw.71) was cultured in serum-free media after which cffDNA was isolated. Trophoblast cells were treated with media, cffDNA (375ng/ml), or as a positive control ODN 2216 (1mM), a synthetic TLR9 agonist. For some experiments, cells were pretreated with A151 (5mM), an antagonist of the DNA sensors TLR9, AIM2, and cGAS, prior to exposure to cffDNA or ODN 2216. Trophoblast cells were also treated with cffDNA or ODN 2216 together with aspirin (ASA, 10mg/ml), aspirin-triggered lipoxin (ATL, 100nM), or hydroxychloroquine (HCQ, 1mg/ml). After 48hrs, cell viability and cell migration were measured using the CellTiter assay and a two-chamber colorimetric assay.

Results: cffDNA and ODN 2216 significantly reduced trophoblast migration by 36.9±4.3% and 47.2±3.2%, respectively (p < 0.05), without affecting cell viability. Reduced cell migration in response to cffDNA or ODN 2216 was significantly reversed by A151 (p < 0.05). cffDNA inhibition of migration was significantly reversed by HCQ, but not ASA or ATL. ODN 2216 inhibition of migration was reversed by HCQ, ASA and ATL (p < 0.05).

Conclusion: Our findings suggest that cffDNA exerts a local effect on placental function by limiting trophoblast migration through activation of DNA sensors. HCQ, which can inhibit TLR9 and cGAS, reverses this, while ASA, which can inhibit cGAS, does not. Thus, cffDNA inhibits trophoblast migration via TLR9. Further research can aid our understanding of the pathogenesis of preeclampsia to improve perinatal outcomes.

Primary & Presenting Author(s)

Daisy Leon-Martinez, MD (she/her/hers)
Assistant Professor
University of California, San Francisco (UCSF)
New Haven, Connecticut, United States

Coauthor(s)

VA
Vikki M. Abrahams, PhD
Yale University School of Medicine
New Haven, Connecticut, United States