Toll-like receptor 9, maternal cell-free DNA and myometrial cell response to CpG oligodeoxynucleotide stimulation

Abstract

Problem: Among mechanisms triggering onset of parturition, it has been recently postulated that Toll-Like Receptor (TLR)9 engagement by cell-free DNA (cfDNA) triggers inflammation, myometrial contractions, and labor in absence of infection. The current study evaluated whether direct (myometrial) or indirect (decidual) TLR9 engagement enhances human myometrial contractility.

Method of study: Toll-like receptor 9 expression and cellular localization were surveyed by immunohistochemistry of placenta, fetal membranes, and myometrium in term (gestational age [GA]: >37 weeks) labor (TL, n = 7) or term non-labor (TNL, n = 7) tissues. Non-pregnant myometrium (n = 4) served as reference. TLR9 mRNA expression relative to other TLRs was evaluated through the mining of an RNA-seq dataset and confirmed by RT-PCR. Immortalized human myometrial cells (hTERT-HM) were treated with incremental concentrations of TLR9 agonist ODN2395, TNF-α, or LPS. Secreted cytokines were quantified by multiplex immunoassay, and contractility was assessed by an in-gel cell contraction assay (n = 9). Induction of hTERT-HM contractility was also evaluated indirectly following exposure to conditioned media from primary term decidual cells (n = 4) previously stimulated with ODN2395.

Results: Toll-like receptor 9 immunostaining in placenta and amniochorion was strongest in decidual cells, but unrelated to labor. TLR9 staining intensity was significantly decreased in TL compared with TNL myometrium (P = 0.002). Although total cfDNA in maternal circulation increased in TL (P = 0.025 vs TNL), difference in cffDNA was non-significant. Myometrial TLR9 mRNA levels were unaffected by contractile status and far less abundant than other pro-inflammatory TLRs. hTERT-HM contractility was enhanced by LPS (P = 0.002) and TNF-α (P = 0.003), but not by ODN2395 (P = 0.345) or supernatant of TLR9-stimulated decidual cells.

Conclusion: Myometrial and decidual TLR9 are unlikely to directly regulate human parturition.

Keywords: DNA; cytokines; decidua; labor; myometrium; toll-like receptor 9.

FIGURE 1.

Presence and localization of TLR9 in human myometrium, fetal membrane and placenta. Representative micrographs of human myometrium (A) term non-labor (TNLm n=7), (B) term labor (TL, n=7), and (C) non-pregnant, immunohistochemically stained for TLR9. (D) Results of semi-quantitative scoring of myometrial (MYO) staining intensity (H-Score). Bars represent mean and standard error. Statistical comparison was conducted using 1-way ANOVA followed by Shapiro-Wilk post-hoc tests. Bars marked with symbols are statistically different at P<0.05 (1-way ANOVA with Holms-Sidak post-hoc tests). Representative micrographs of full thickness fetal membranes (FM) from (E) TNL and (F) TL deliveries with matched sections of placenta (G) TNL and (H) TL. Photographs were taken at 100× magnification for panels (scale bar: 100 nm) and 400× for insets (scale bar 25 nm). ae: amnion epithelium; am: amnion; cd: choriodecidua; de: decidual cells; pv: placental villi.

FIGURE 2.

Human myometrial TLR9 expression by RT-PCR, relative expression to other TLRs by RNA-seq, and serum cfDNA and cffDNA levels in maternal circulation. (A) Relative quantitation (RQ) by real-time RT-PCR of TLR9 mRNA expression in myometrium and villous placenta relative to expression of housekeeping genes (HKG). (B) mRNA expression by RNA sequencing (RNA-seq) for the 10 known TLRs (TLR1-TLR10) in term non-labor (TNL, n=5) and labor (TL, n=5) myometrium. (C) Genomic equivalents of total cell-free DNA (cfDNA) and (D) cell-free fetal DNA (cffDNA) in maternal circulation in term women not in labor (TNL) or in active labor (TL). Results are shown as mean and standard error. The asterisk (*) denotes statistically significant difference at P<0.05 (Student t-test).

FIGURE 3.

Effects of TLR stimulation on cytokine secretion by hTERT-HM cells. Time-course of IL-6 concentration in culture medium after challenge with (A) increasing concentrations of the TLR9 agonist ODN2395 (0.5, 1, or 2 μM) or (B) LPS (5 μg/mL). At each time point and for each independent experiment (n=4), IL-6 concentrations in wells with ODN2395 were normalized to IL-6 concentration in wells treated with the control oligonucleotide (ODN-CRL, has GpC nucleotides instead of CpG) and illustrated by the dashed horizontal line marking 100% baseline level. IL-6 concentrations in wells with LPS were normalized to levels measured in wells treated with the equivalent volume of endotoxin-free water (vehicle for LPS) which represents their baseline. *P<0.05 vs. baseline †P<0.05 vs. t=0 hours (T0), 2-way repeated measures ANOVA followed by post-hoc Holms-Sidak tests. (C) Concentration of a panel of cytokines measured by multiplex immunoassay in medium of hTERT-HM cells collected after 24 hours stimulation with low dose, 0.5 μM) or (D) high dose (2 μM) of TLR agonist ODN2395. (E) Cytokine levels after stimulation with LPS (5 μg/mL). Data is shown as mean and standard error. *P<0.05 vs. baseline (1-way repeated measures ANOVA with correction for multiple comparisons using the Holms-Sidak method).

FIGURE 4.

Effect of TLR stimulation on contractility of hTERT-HM cells embedded in collagen gels. (A) Representative gel contraction assay after 24 h treatment with water (vehicle), LPS (5 μg/mL) or ODN2395 (1 μM). (B) Results of collagen gel contraction assays after treatment with the TLR9 agonist ODN2395 (1 μM), LPS (5 μg/mL), TNF-α (10 ng/mL). Data was normalized to control wells which received water diluent and represent a common baseline for each independent experiment (n=6). (C) Results of collagen gel contraction assay after exposure to TLR9 agonist ODN2395 (1 μM), TLR9 antagonist A151 (1 μM), or their combination. Data was normalized to equivalent gels in each independent experiment (n=3) treated with ODN-CRL and considered as baseline. Data is shown as mean and standard error. *P<0.05 vs. baseline †P<0.05 vs. t=0 hours (T0), 2-way repeated measures ANOVA followed by post-hoc Holms-Sidak tests to correct for multiple comparisons.

FIGURE 5.

TLR9-mediated effects on oxytocin-induced contractility and decidual cell condition medium (DCM) on hTERT-HM cells embedded in collagen gels. (A) Contractility of hTERT-HM cells was enhanced by exposure to 100 ng/mL but not by 10 ng/mL of oxytocin. Data was normalized to gels treated with endotoxin-free water. (B) Effect of ODN2395 (CpG ODN) and ODN-CRL (GpC ODN) in the presence of 100 ng/mL oxytocin. Baseline reflects area of equivalent gels treated with 100 ng/mL oxytocin which was used for normalization for each individual experiment (n=4). (C) Indirect effects of decidual cell TLR9 stimulation on contraction of hTERT-HM cells embedded in collagen gels. Primary decidual cells were treated in culture for 1 μM ODN2395 or ODN-CRL (GpC-ODN, 1 μM) and DCM collected at 24 hours. Collagen gels with embedded hTERT-HM cells were then exposed to DCM for up to 24 hours. Percent gel contraction was normalized to baseline representing gels incubated in conditioned medium from decidual cells treated with equivalent volume of endotoxin-free water. The entire experiment was repeated 3 times. Data is shown as mean and standard error. *P<0.05 relative to baseline; †P<0.05 relative to t=0 hours (T0); ‡P<0.05 vs. ODN-CRL (2-way repeated measures ANOVA followed by post-hoc Holms-Sidak tests).