Fetal and Placental DNA Stimulation of TLR9: A Mechanism Possibly Contributing to the Pro-inflammatory Events During Parturition

Abstract

Introduction:

While there is evidence for a relationship between cell-free fetal DNA (cffDNA) and parturition, questions remain regarding whether cffDNA could trigger a pro-inflammatory response on the pathway to parturition. We hypothesized that placental and/or fetal DNA stimulates toll-like receptor 9 (TLR9) leading to secretion of pro-inflammatory cytokines by macrophage cells.

Methods:

Four in vitro DNA stimulation studies were performed using RAW 264.7 mouse peritoneal macrophage cells incubated in media containing the following DNA particles: an oligodeoxynucleotide (ODN2395), intact genomic DNA (from mouse placentas, fetuses and adult liver), mouse DNA complexed with DOTAP (a cationic liposome forming compound), and telomere-depleted mouse DNA. Interleukin 6 (IL6) secretion was measured in the media by enzyme-linked immunosorbent assay; and the cell pellet was homogenized for protein content (picograms IL6/mg protein).

Results:

Robust IL6 secretion was observed in response to ODN2395 (a CpG-rich TLR9 agonist), mouse DNA-DOTAP complexes, and telomere-depleted mouse DNA in concentrations of 5 to 15 μg/mL. In contrast, ODN A151 (containing telomere sequence motifs), intact genomic mouse DNA, and restriction enzyme-digested DNA had no effect on IL6 secretion. The IL6 response was significantly inhibited by chloroquine (10 μg/mL), thereby confirming the important role for TLR9 in the response by macrophage cells.

Conclusions:

DNA derived from mouse placentas and fetuses, and depleted of telomeric sequences, stimulates a robust pro-inflammatory response by macrophage cells, thereby supporting the hypothesis that cffDNA is able to stimulate an innate immune response that could trigger the onset of parturition. These findings are of clinical importance, as we search for effective treatment/prevention of preterm parturition.

Keywords

fetal cell-free DNA placental DNA mouse macrophage cells telomeres toll-like receptor 9

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References

Kelly

. Inflammatory mediators and parturition. Rev Reprod. 1996;1(2):89–96.

Lindström

Bennett

. The role of nuclear factor kappa B in human labour. Reproduction. 2005;130(5):569–581.

Patni

Flynn

Wynen

. An introduction to Toll-like receptors and their possible role in the initiation of labour. BJOG. 2007;114(11):1326–1334.

Phillippe

. Cell-free fetal DNA, telomeres, and the spontaneous onset of parturition. Reprod Sci. 2015;22(10):1186–1201.

Tein

Lau

. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet. 1998;62(4):768–775.

Chan

Zhang

Hui

. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem. 2004;50(1):88–92.

Zhang

Leung

Lau

Chang

Hjelm

. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet. 1999;64(1):218–224.

Sun

Qin

Shan

. Differential effects of the CpG-Toll-like receptor 9 axis on pregnancy outcome in nonobese diabetic mice and wild-type controls. Fertil Steril. 2013;99(6):1759–1767.

Ariga

Ohto

Busch

. Kinetics of fetal cellular and cell-free DNA in the maternal circulation during and after pregnancy: implications for noninvasive prenatal diagnosis. Transfusion. 2001;41(12):1524–1530.

10.

Birch

English

O’Donoghue

Barigye

Fisk

Keer

. Accurate and robust quantification of circulating fetal and total DNA in maternal plasma from 5 to 41 weeks of gestation. Clin Chem. 2005;51(2):312–320.

11.

Majer

Bauer

Magnet

. Maternal urine for prenatal diagnosis–an analysis of cell-free fetal DNA in maternal urine and plasma in the third trimester. Prenat Diagn. 2007;27(13):1219–1223.

12.

Farina

LeShane

Romero

. High levels of fetal cell-free DNA in maternal serum: a risk factor for spontaneous preterm delivery. Am J Obstet Gynecol. 2005;193(2):421–425.

13.

Jakobsen

Clausen

Rode

Dziegiel

Tabor

. High levels of fetal DNA are associated with increased risk of spontaneous preterm delivery. Prenat Diagn. 2012;32(9):840–845.

14.

Leung

Zhang

Lau

Hjelm

. Maternal plasma fetal DNA as a marker for preterm labour. Lancet. 1998;352(9144):1904–1905.

15.

Caughey

Stotland

Washington

Escobar

. Who is at risk for prolonged and postterm pregnancy?. Am J Obstet Gynecol. 2009;200(6):683.e1-683.e5.

16.

Heslehurst

Simpson

Ells

. The impact of maternal BMI status on pregnancy outcomes with immediate short-term obstetric resource implications: a meta-analysis. Obes Rev. 2008;9(6):635–683.

17.

Wang

Batey

Struble

Musci

Song

Oliphant

. Gestational age and maternal weight effects on fetal cell-free DNA in maternal plasma. Prenat Diagn. 2013;33(7):662–666.

18.

Scharfe-Nugent

Corr

Carpenter

. TLR9 provokes inflammation in response to fetal DNA: mechanism for fetal loss in preterm birth and preeclampsia. J Immunol. 2012;188(11):5706–5712.

19.

Gursel

Yamada

Ishii

Takeshita

Klinman

. Repetitive elements in mammalian telomeres suppress bacterial DNA-induced immune activation. J Immunol. 2003;171(3):1393–1400.

20.

Thaxton

Romero

Sharma

. TLR9 activation coupled to IL-10 deficiency induces adverse pregnancy outcomes. J Immunol. 2009;183(2):1144–1154.

21.

Kuznik

Bencina

Svajger

Jeras

Rozman

Jerala

. Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines. J Immunol. 2011;186(8):4794–4804.

22.

Yasuda

Ogawa

Yamane

Nishikawa

Takakura

. Macrophage activation by a DNA/cationic liposome complex requires endosomal acidification and TLR9-dependent and -independent pathways. J Leukoc Biol. 2005;77(1):71–79.

23.

Kipling

Cooke

. Hypervariable ultra-long telomeres in mice. Nature. 1990;347(6291):400–402.

24.

Sackesen

van de Veen

Akdis

. Suppression of B-cell activation and IgE, IgA, IgG1 and IgG4 production by mammalian telomeric oligonucleotides. Allergy. 2013;68(5):593–603.

25.

Shirota

Gursel

Klinman

. Suppressive oligodeoxynucleotides inhibit Th1 differentiation by blocking IFN-gamma- and IL-12-mediated signaling. J Immunol. 2004;173(8):5002–5007.

26.

Hemmi

Takeuchi

Kawai

. A Toll-like receptor recognizes bacterial DNA. Nature. 2000;408(6813):740–745.

27.

Krieg

Matson

. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. 1995;374(6522):546–549.

28.

Kumagai

Takeuchi

Akira

. TLR9 as a key receptor for the recognition of DNA. Adv Drug Deliv Rev. 2008;60(7):795–804.

29.

Pisetsky

Reich

. Inhibition of murine macrophage IL-12 production by natural and synthetic DNA. Clin Immunol. 2000;96(3):198–204.

30.

Dong

Ito

Ishii

Klinman

. Suppressive oligonucleotides protect against collagen-induced arthritis in mice. Arthritis Rheum. 2004;50(5):1686–1689.

31.

Panter

Kuznik

Jerala

. Therapeutic applications of nucleic acids as ligands for Toll-like receptors. Curr Opin Mol Ther. 2009;11(2):133–145.

32.

Yasuda

Kirschning

. Endosomal translocation of vertebrate DNA activates dendritic cells via TLR9-dependent and -independent pathways. J Immunol. 2005;174(10):6129–6136.

33.

Norman

Bollapragada

Yuan

Nelson

. Inflammatory pathways in the mechanism of parturition. BMC Pregnancy Childbirth. 2007;7(suppl 1):S7.

34.

Edey

O’Dea

Herbert

. The Local and Systemic Immune Response to Intrauterine LPS in the Prepartum Mouse. Biol Reprod. 2016;95(6):125.

35.

Takagi

Hashiguchi

Mahato

Tokuda

Takakura

Hashida

. Involvement of specific mechanism in plasmid DNA uptake by mouse peritoneal macrophages. Biochem Biophys Res Commun. 1998;245(3):729–733.

36.

Krieg

Weeratna

. Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs. Proc Natl Acad Sci U S A. 1998;95(21):12631–12636.

37.

Zeuner

Ishii

Lizak

. Reduction of CpG-induced arthritis by suppressive oligodeoxynucleotides. Arthritis Rheum. 2002;46(8):2219–2224.

38.

Smith

Baker

Symonds

. Placental apoptosis in normal human pregnancy. Am J Obstet Gynecol. 1997;177(1):57–65.

39.

Smith

Baker

. Placental apoptosis is increased in post-term pregnancies. Br J Obstet Gynaecol. 1999;106(8):861–862.

40.

McLaren

Taylor

Bell

. Increased incidence of apoptosis in non-labour-affected cytotrophoblast cells in term fetal membranes overlying the cervix. Hum Reprod. 1999;14(11):2895–900.

41.

Imaeda

Watanabe

Sohail

. Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome. J Clin Invest. 2009;119(2):305–314.

42.

Watanabe

Hashmi

Gomes

. Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9. Hepatology. 2007;46(5):1509–1518.

43.

Multani

Ozen

Narayan

. Caspase-dependent apoptosis induced by telomere cleavage and TRF2 loss. Neoplasia. 2000;2(4):339–345.

44.

Nadeau-Vallee

Obari

. Sterile inflammation and pregnancy complications: a review. Reproduction. 2016;152(6):R277–R292.