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Preeclamptic patient-derived circulating cell-free DNA activates the production of inflammatory cytokines via toll-like receptor 9 signalling in the human placenta

Ozeki, Ayaea; Tani, Kumikoa; Takahashi, Hironorib; Suzuki, Hirotadab; Nagayama, Shihob; Hirashima, Chikakob; Iwata, Hisatakaa; Kuwayama, Takehitoa; Ohkuchi, Akihideb; Shirasuna, Koumeia

doi: 10.1097/HJH.0000000000002208

Objectives: Preeclampsia, a pregnancy-specific syndrome, is associated with maternal systemic and placental inflammatory responses. Cell-free DNA (cfDNA) and cf-foetal DNA (cffDNA) in the blood are elevated in patients with preeclampsia and act as danger signals. Placenta-derived foetal DNA induces inflammatory responses and pregnancy complications in mice. However, whether extracellular DNA from the placenta really causes inflammatory responses remains unclear. Therefore, we investigated the effect of serum cfDNA and placental cffDNA on inflammatory responses using normal pregnant women and preeclampsia patients.

Methods: Sera were taken from normal pregnant women and preeclampsia patients, and human trophoblast cell line Sw.71 cells were treated with serum with or without toll-like receptor 9 (TLR9; a sensor of exogenous DNA) inhibitor and genome elimination reagent. For cffDNA collection, placental tissue from the participants was cultured, and the released cffDNA was administrated to Sw.71 cells.

Results: The amount of serum cfDNA was higher in preeclampsia patients than in normal pregnant women. Treatment of preeclampsia serum stimulated inflammatory cytokine secretion, which was inhibited by a genome elimination reagent. Expression levels of TLR9 and amount of cffDNA from the placenta were higher in preeclampsia patients than of normal pregnant women. Preeclampsia-derived cffDNA increased inflammatory cytokine levels compared with normal pregnant derived cffDNA.

Conclusion: In human trophoblast cells, preeclampsia patient-derived cfDNA increased inflammatory cytokine levels via TLR9. Preeclampsia placenta released more cffDNA, which stimulated inflammatory cytokine. We suggest that elevated circulating cfDNA and cffDNA induces placental inflammatory responses, resulting in accelerated pathological features of preeclampsia.

aLaboratory of Animal Reproduction, Department of Animal Science, Tokyo University of Agriculture, Atsugi, Kanagawa

bDepartment of Obstetrics and Gynecology, Jichi Medical University, Shimotsuke, Tochigi, Japan

Correspondence to Koumei Shirasuna, PhD, Laboratory of Animal Reproduction, Department of Animal Science, Tokyo University of Agriculture, 1737 Funako, Atsugi, Kanagawa 234-0034, Japan. Tel: +81 46 270 6588; fax: +81 46 247 4338; e-mail:

Abbreviations: cfDNA, cell-free DNA; cffDNA, cf-fetal DNA; cGAS, protein cyclic GMP-AMP synthase; DAMP, danger-associated molecular pattern; HUVEC, human umbilical vein endothelial cells interleukin; IFI16, interferoninducible protein 16; IFN, interferon; IL, interleukin; LDH, lactate dehydrogenase; sEng, soluble endoglin; sFlt1, soluble fms-like tyrosine kinase; STING, the stimulator of interferon genes; TLR9, toll-like receptor

Received 18 December, 2018

Revised 27 April, 2019

Accepted 10 July, 2019

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