Concentrations and immunostimulatory potential of circulating cell-free membrane-bound and membrane-unbound mitochondrial DNA in preeclampsia

Abstract

Cell-free circulating mitochondrial DNA (CFCmtDNA) is a damage-associated molecular pattern (DAMP) that can activate Toll-like receptor 9 (TLR-9). The main objectives of this case-control study were 1) to determine absolute concentrations and immunostimulatory capacity of CFCmtDNA, in membrane-bound and -unbound states, in cases with preeclampsia and healthy controls and 2) to implement a bootstrapped penalized regression analysis to establish the contribution of CFCmtDNA to preeclampsia diagnosis and its interaction with commonly collected patient characteristics. To determine the contribution of membrane-bound and -unbound CFCmtDNA in preeclampsia, DNA from plasma samples was exctracted with lysis buffer (membrane-unbound) and without lysis buffer (membrane-bound). CFCmtDNA, quantified using absolute PCR quantification protocol, was reduced in preeclampsia compared to healthy controls (P≤0.02). While the pattern of reduced CFCmtDNA in preeclampsia was similar between methods of DNA extraction, DNA isolation with membrane lysis buffer resulted in 1,000-fold higher CFCmtDNA quantification in the preeclampsia group (P=0.0014) and 430-fold higher CFCmtDNA quantification in the control group (P<0.0001). Even though CFCmtDNA concentrations were reduced, plasma from women with preeclampsia induced greater TLR-9 activation than plasma from gestational age matched controls (P≤0.01) as monitored using SEAP reporter 293 cells expressing human TLR-9. Penalized regression analysis showed that women with preeclampsia are strongly likely to have high concentrations of nDNA and DNase I along with a prior history of preeclampsia. Low concentrations of CFCmtDNA and mode of delivery were also associated with preeclampsia. In conclusion, our data demonstrate increases in the immunostimulatory potential of CFCmtDNA and upregulation of DNA degradation mechanisms in women with preeclampsia at the third trimester.

Mitochondria-mediated Maternal-fetal Interactions and Consequences of Mitochondrial Dysregulation Indicate New Roles for Mitochondria in Hypertensive Pregnancies

“…As placenta-derived EVs are more abundant in pregnancies with preeclampsia [29], our findings suggest a direct consequence for fetal mitochondrial dysregulation in cell-cell communication, and possible delivery of placental factors that can affect maternal physiology. Further, our work has shown ccf-mtDNA primarily exist in a membranebound state in both normotensive mothers and mothers with preeclampsia [4], and others have shown the presence of sFLT1 in placenta-derived EVs [30,31]. If this pattern is present throughout pregnancy, it may implicate fetal inheritance of mitochondrial dysregulation as an important contributor to pregnancy-induced hypertension, particularly as it pertains to placental function and communication between mother and fetus.…”

“…However, consequences of mitochondrial dysfunction in hypertensive pregnancies are not fully described, nor is the role of maternal versus fetal mitochondrial dysfunction clearly delineated. In support of the hypothesis that mitochondrial dysfunction underlies pregnancy-induced hypertension, we recently demonstrated impaired circulating cell-free mitochondrial DNA (ccf-mtDNA) dynamics in pregnant mothers with preeclampsia [4]. This finding is highly relevant to preeclampsia pathophysiology, as ccf-mtDNA is often used as a noninvasive proxy for cellular stress and systemic inflammation due to its ability to activate Toll-like receptor 9 and other DNA-sensing receptors [5,6].…”

“…Mutational load analysis was carried out using whole mitochondrial genome amplification followed by deep Illumina-based next generation sequencing. Subject characteristics and sample collection are detailed in [4]. Sample collection occurred during the 3 rd trimester (28 -41 weeks of gestation).…”

Gestational exposure to unmethylated CpG oligonucleotides dysregulates placental molecular clock network and fetoplacental growth dynamics, and disrupts maternal blood pressure circadian rhythms in rats

“…The copyright holder for this preprint this version posted March 15, 2023. ; https://doi.org/10.1101/2023.03.14.532649 doi: bioRxiv preprint placentas and circulation of pregnant women diagnosed with pregnancy complications such as preeclampsia (11,(68)(69)(70), and this TLR9 dysregulation may alter the impact of CpG ODN exposure on immune modulation during pregnancy. Importantly, we and others have previously demonstrated the pregnancy-specific effects of CpG ODN exposure on perinatal outcomes (37, 39).…”

Oxidative stress induces release of mitochondrial DNA into the extracellular space in human placental villous trophoblast BeWo cells

“…In preeclampsia, a severe hypertensive disorder of pregnancy with placental pathology, ccf-mtDNA concentrations differ from healthy controls. Some studies have reported an increase (7,8) and others showed a reduction (9,10) in maternal ccf-mtDNA in patients with preeclampsia, depending on gestational age and method of mtDNA quantification (11).…”