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It has been proposed that cell-free fetal DNA (derived from the placenta) stimulates TLR9 thereby activating an innate immune response within the pregnant uterus, thus triggering spontaneous parturition. Vertebrate DNA is normally a poor agonist for TLR9; however, removal of telomere sequences, as occurs during cellular apoptosis, reverses this negative effect. These studies sought to test the hypothesis that placental DNA is able to stimulate a robust proinflammatory cytokine response after enzymatic cleavage of telomere regions.
Genomic DNA was obtained from maternal, fetal and placental tissues from pregnant CD-1 mice (DNA extraction kit from Roche). The DNA was then incubated with the exonuclease BAL-31 (which cleaves the telomere ends of the chromosomal DNA). The BAL-31 treated DNA was incubated with mouse peritoneal macrophages (RAW 264.7 cells from ATCC) to assess TLR9 stimulation. Positive control incubations were performed using ODN2395 (a TLR9 agonist). IL6 concentration in the incubation media was assayed by ELISA (BioLegend), normalized for cellular protein, and reported as picograms (pg) per milligram (mg) protein. Each experiment N = 4.
The optimal BAL-31 digestion time was 2 hours. The maximal stimulation occurred with 1 - 5 mcg/mL BAL-31 treated placental DNA (result = 11,431 - 15,140 pg IL6/mg protein); results significantly (p< 0.01) higher than found with untreated DNA (= 5.7 pg/mg) or ODN2395 (= 944 pg/mg). Timed studies demonstrated increased IL6 at 6 hours which peaked at 24 hours after the DNA addition. Compared to placental DNA, BAL-31 treated fetal DNA produced a similar robust IL6 response (= 13,654 pg/mL), whereas BAL-31 treated DNA from maternal liver produced a significantly lower IL6 response (= 6,313 ng/mg, p< 0.01).
During apoptosis, DNA undergoes degradation, including removal of telomere sequences. These studies have demonstrated that placental DNA depleted of telomeres stimulates robust IL6 production by macrophage cells. Inflammation appears to plays a key role during the onset of spontaneous parturition; these studies have provided additional support for the hypothesis that cell-free fetal DNA, generated during placental apoptosis, is able to trigger these events. (Funded by VCRB at MGH)