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. Author manuscript; available in PMC: 2015 May 1.
Published in final edited form as: Am J Obstet Gynecol. 2013 Nov 28;210(5):445.e1–445.e6. doi: 10.1016/j.ajog.2013.11.037

Longitudinal expression of Toll-like receptors on dendritic cells in uncomplicated pregnancy and postpartum

Brett C Young 1, Aleksandar K Stanic 1, Britta Panda 1, Bo R Rueda 1, Alexander Panda 1
PMCID: PMC4374641  NIHMSID: NIHMS580291  PMID: 24291497

Abstract

OBJECTIVE

Toll-like receptors (TLRs) are integral parts of the innate immune system and have been implicated in complications of pregnancy. The longitudinal expression of TLRs on dendritic cells in the maternal circulation during uncomplicated pregnancies is unknown. The objective of this study was to prospectively evaluate TLRs 1-9 as expressed on dendritic cells in the maternal circulation at defined intervals throughout pregnancy and postpartum.

STUDY DESIGN

This was a prospective cohort of 30 pregnant women with uncomplicated pregnancies and 30 nonpregnant controls. TLRs and cytokine expression was measured in unstimulated dendritic cells at 4 defined intervals during pregnancy and postpartum. Basal expression of TLRs and cytokines was measured by multicolor flow cytometry. The percent-positive dendritic cells for each TLRs were compared with both nonpregnant and postpartum levels with multivariate linear regression.

RESULTS

TLRs 1, 7, and 9 were elevated compared with nonpregnant controls with persistent elevation of TLR 1 and interleukin-12 (IL-12) into the postpartum period. Concordantly, levels of IL-6, IL-12, interferon alpha, and tumor necrosis factor alpha increased during pregnancy and returned to levels similar to nonpregnant controls during the postpartum period. The elevated levels of TLR 1 and IL-12 were persistent postpartum, challenging notions that immunologic changes during pregnancy resolve after the prototypical postpartum period.

CONCLUSION

Normal pregnancy is associated with time-dependent changes in TLR expression compared with nonpregnant controls; these findings may help elucidate immunologic dysfunction in complicated pregnancies.

Keywords: dendritic cells, innate immune system, pregnancy, toll-like receptors

The maternal immune system plays an integral role at the maternal-fetal interface;1–8 yet all the specific contributions of the immune system in normal and abnormal pregnancies remain to be elucidated. Toll-like receptors (TLRs) are an important aspect of the innate immune system that recognize both microbial and endogenous ligands as well as host products released during tissue damage.1,9 Much evidence exists to support a role for TLRs in both uncomplicated pregnancies2,8 and pregnancies complicated by preeclampsia,6,10 and preterm labor.11–13 However, there is limited information on whether expression of TLRs in the maternal circulation changes during uncomplicated pregnancies. By better understanding any time-dependent changes in TLR in normal pregnancies, comparisons can be made to TLR levels in pathologic conditions of pregnancy such as preterm labor, preeclampsia, and stillbirth.

TLRs are expressed on various antigen-presenting cells; dendritic cells are a main group of antigen-presenting cells that express 9 of the 10 TLR isoforms expressed in humans. There are 2 types of dendritic cells that differ in their TLR expression: myeloid dendritic cells typically express TLRs 1-6 and 8, whereas plasmacytoid dendritic cells express TLRs 7 and 9.14 TLRs bind to highly conserved protein sequences known as pathogen-associated molecular patterns (PAMPs), which are expressed by and unique to specific microorganisms or endogenous ligands.15 TLR 1 binds triacetylated lipoproteins, a component of gram-positive bacteria. TLR 2 recognizes bacterial lipoproteins, gram-positive bacterial peptidoglycan and lipoteichoic acid through the formation of heterodimers with TLR 1 or TLR 6. TLR 3 binds double-stranded RNA, and TLR 4 binds gram-negative bacterial lipopolysaccharide. TLR 5 recognizes bacterial flagellin; TLR 6 binds diacylated lipoprotein. TLR 7 and TLR 8 bind single-stranded RNA. TLR 9 binds nonmethylated CpG DNA, including fetal DNA.16 In addition, TLRs interact with endogenous molecules called danger-associated molecular patterns (DAMPs) including reactive oxygen species and proteins released from dying cells under stress.1,17 For example, TLR 4 and TLR 2 can bind DAMPs, such as heat shock protein 60, heat shock protein 70, and fibrinogen.1

Recent studies demonstrate a significant role of specific TLRs in both normal and complicated pregnancies at the maternal-fetal interface throughout gestation.1–8 Elucidation of normative trends in these pregnancies may promote further understanding of altered TLR expression in women with complicated pregnancies.

In this current study, we sought to evaluate longitudinal TLR expression in dendritic cells during normal term pregnancies compared with the post-partum state and to nonpregnant controls. On the basis of prior studies demonstrating a proinflammatory state in the third trimester, we hypothesized that TLR and cytokine expression will increase toward the third trimester and return to baseline levels at the time of the postpartum collection.

Materials and Methods

Subject recruitment

After receiving institutional review board approval, we recruited patients receiving care at 2 teaching hospitals. For the pregnant cohort, nonobese women of any parity were included if they had singleton gestations and were without significant medical conditions including diabetes or chronic hypertension. Similarly, we selected control patients through recruitment in the gynecologic clinic after initial screening by the patient’s physician. Women self-identified as free of medical problems and cigarette smoking. Women self-reported their last menstrual periods and whether there was possibility of an undiagnosed pregnancy. Women in both groups denied any use of immune-modulating medications or recent significant illnesses requiring antibiotics. Body mass index was based on self-reported height and scale-obtained weight at the time of the initial visit.

Blood sampling

Blood was obtained at 4 standardized collection times for the cohort of pregnant women: Collection 1 (first trimester) during routine initial prenatal laboratories or at the time of elective genetic screening; Collection 2 (mid-trimester) between 26–28 weeks at the time of routine gestational diabetes screening; Collection 3 (day of delivery) on presentation to labor/delivery before delivery of the infant; Collection 4 (postpartum) at the scheduled 6 week postpartum visit. Enrolled women had additional research blood obtained at the time of routine intravenous placement and standard admission laboratories on presentation to labor/delivery because of scheduled induction of labor, spontaneous labor, or scheduled cesarean delivery. Blood samples from nonpregnant control women were collected at clinic visits.

Isolation of human peripheral blood dendritic cells

Human peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Histopaque (Sigma-Aldrich, St. Louis, MO) gradient centrifugation.

Cell staining and flow cytometry

For fluorescence-activated cell sorting (FACS) assays, dendritic cells were identified in the PBMC preparations by staining with fluorescent antibodies to specific surface markers. PBMCs were suspended in RPMI 1640 medium plus 10% FBS and adjusted to a concentration of 2 × 106 cells/mL. Cells were then washed and frozen in 90% fetal bovine serum (FBS) containing 10% DMSO, and stored at −80°C. On the day of staining and analysis, cells were thawed, and cell surface labeling at 4°C was followed by washing in PBS containing 2% FBS and 0.05% sodium azide. For samples where intracellular staining was performed, they were fixed with BD Cytofix/Cytoperm buffer, then permeabilized in BD Perm/Wash buffer as described in the manufacturer’s protocol.

For analyses of TLR expression, conventional and plasmacytoid dendritic cells were identified using antibodies against cell-surface markers (Anti-CD3 APC-Cy7, -CD14 APC-Cy7, -CD16 APC-Cy7, -CD19 APC-Cy7, -CD11c APC, -CD123 PE-Cy5, -HLA-DR PE-Cy7; BD Pharmingen, San Diego, CA). Cell-surface TLR expression was assessed using antibodies against TLR 1 (PE, clone GD2.F4; eBioscience, San Diego, CA), TLR 2 (Alexa 700, clone TL2.1; eBioscience), TLR 4 (Alexa700, HTA125; eBioscience), TLR 5 (FITC, 85B152.5; AbCam); intracellular TLR expression using antibodies against TLR 3 (FITC, 40C1285.6; AbCam), TLR 7 (FITC, 533707; R&D Systems), TLR 8 (PE, 44C143; AbCam), TLR 9 (PE, eB72-1665; BD Pharmingen); and finally, intracellular cytokine expression was assessed using interleukin-6 (IL-6) (PE, MQ2-6A3; BD Pharmingen), IL-12 (e450, Clone C8.6; eBioscience), tumor necrosis factor alpha (TNFα) (Alexa700, MAb11; BDPharmingen), interferon alpha (IFNα) (FITC, Clone FHC520; Chromaprobe, Maryland Heights, MO).

Approximately 0.2–0.5 million total events per sample were assessed using an LSR II flow cytometry instrument (BD Biosciences) with analysis using FlowJo software (Tree Star, Ashland, OR). Initial gating on parent dendritic cell subsets was further characterized by assessment of TLR and cytokine expression.

Statistical analysis

A sample size calculation was performed to detect a 20% difference in TLR protein expression between the pregnant and nonpregnant cohort with a power of 0.8. Therefore, we aimed to enroll 29 pregnant women and 29 nonpregnant controls. Assuming 30% loss to follow-up from miscarriage or missed collection because of logistical reasons, we aimed to enroll 40 pregnant women.

Pregnant women with 4 sample collections were included for analysis. Where appropriate, proportions or means were used to describe the demographic and clinical characteristics of each cohort at enrollment. Test of normality (Shapiro-Wilks) was applied to analyze the datasets; all data satisfied criteria for a normal distribution. We used multivariate linear regression to estimate the effect of pregnancy on the percent-positive cells expressing specific TLR proteins; results are shown as the mean difference of percent positive cells for each gated TLR or cytokine compared with both the nonpregnant control group and the postpartum collection. We controlled for confounding by covariates (maternal age, body mass index, and parity). A mixed-effects model was used to model both the variation in our sample of nonpregnant and pregnant subjects as well as the correlation with repeated ligand-specific stimulation, which may increase Type 1 error through family-wise multiple comparison. In our final model, we used an unstructured covariance structure that permitted each participant to have a unique correlation structure. This model accounted for the issues of a heterogenous cohort and the cross-sectional repeated measurement of TLR and cytokine outcomes by using the Hochberg multiple comparison procedure. Statistical tests were 2-tailed and P < .05 was considered statistically significant. SAS version 9.1 (SAS Institute, Cary, NC) was used for analyses.

Results

TLR and cytokine expression on dendritic cells from 30 pregnant women was analyzed at predefined intervals; similarly, TLR and cytokine expression was analyzed from 30 nonpregnant women (controls). Of the initial 40 pregnant women followed prospectively, 1 woman experienced a first trimester miscarriage and 9 other women missed 1 or more collection samples because of scheduling issues. Therefore, 150 samples from 30 pregnant and 30 nonpregnant women were analyzed. Samples from the first trimester (collection 1), third trimester (collection 2), and before delivery (collection 3) were collected at 11.0 ± 2.2 weeks; 27.1 ± 1.2 weeks, and 39.8 ± 1.1 weeks, respectively. Eleven women delivered via cesarean delivery and the majority (76.7%) of women experienced labor before the collection 3 blood draw. The postpartum sample (collection 4) was collected 6.9 ± 1.1 weeks after delivery. The clinical demographics of the cohort are displayed in Table 1. The cohort showed pregnant subjects were more likely to be older and parous. No pregnant woman developed preeclampsia. One patient developed mild range gestational hypertension without proteinuria. There were no postterm (>42 week) deliveries. One patient developed PPROM at 35.9 weeks’ gestation and was induced. There were no cases of fetal growth restriction. The mean birth-weight was 3511 g.

TABLE 1.

Clinical demographics

Characteristic Pregnant women (n = 30) Nonpregnant women (n = 30) Significance (P value)
Maternal age, y 32.2 ± 5.0 24.5 ± 1.9 .0001
BMI, kg/m2 22.6 ± 3.0 21.5 ± 1.2 NS
White, % 97.1 96.7 NS
Gravidity 2.1 ± 1.5 0.03 ± 0.2 .0001
Parity 0.65 ± 1.30 0.03 ± 0.2 .009
Smoking, % 3.3 0 NS
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Results expressed as mean ± standard deviation or proportions where indicated.

BMI, body mass index; NS, nonsignificant.

We detected longitudinal trends of select TLRs in both myeloid and plasmacytoid dendritic cells compared with both nonpregnant controls and the postpartum sample. As shown in Figure 1, compared with nonpregnant women, pregnant women had statistically higher myeloid dendritic cell expression of TLR 1 throughout pregnancy. Similarly, dendritic cells of pregnant women had statistically higher basal expression of TLR 7 and TLR 9 during the first trimester, early third trimester and before delivery. Post-partum, there was a continued elevation in percent-positive dendritic cells for TLR 1 compared with both the non-pregnant controls and the first trimester sample. Similarly, TLR 7 expression remained elevated postpartum compared with nonpregnant controls but was statistically lower than the first trimester collection. There was no statistical change in the relative level of TLRs 2/6, 3, 4, 5, or 8 throughout pregnancy compared with nonpregnant controls or the postpartum sample (Figure 2).

FIGURE 1. Longitudinal expression of select TLRs and cytokines during pregnancy and postpartum: significant trends.

FIGURE 1

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Longitudinal basal expression of TLRs 1, 7, and 9 and cytokines IL-12, IL-6, and TNFα in normal pregnancies and postpartum shown as the mean difference of percent positive cells for each gated TLR or cytokine compared with both the nonpregnant control group and the postpartum collection.

IL, interleukin; TLRs, Toll-like receptors; TNFα, tumor necrosis factor alpha.

*P < .05 compared with nonpregnant controls; #P < .05 compared with postpartum collection.

FIGURE 2. Longitudinal expression of select TLRs during pregnancy and postpartum: nonsignificant trends.

FIGURE 2

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Longitudinal basal expression of TLRs 2/6, 3, 4, 5, 8 in normal pregnancies and postpartum shown as the mean difference of percent positive cells for each gated TLR or cytokine compared with both the nonpregnant control group and the postpartum collection. There were no statistical significant differences.

TLRs, Toll-like receptors.

Compared with nonpregnant controls, myeloid dendritic cells in pregnant women expressed higher levels of cytokines IL-6 at each collection point during pregnancy that subsequently decreased at the time of the postpartum collection to levels similar to the nonpregnant state. IL-12 increased throughout pregnancy and remained elevated postpartum compared with nonpregnant controls. Expression of TNFα in myeloid and plasmacytoid dendritic cells remained increased throughout pregnancy and decreased postpartum similar to nonpregnant controls. In plasmacytoid dendritic cells, IFNα similarly increased throughout pregnancy compared with both nonpregnant controls and post-partum and then decreased in the puerperium to levels similar to the nonpregnant state (Figure 1).

Comment

Recent research implicates immune system involvement in the biology of normal pregnancies and the pathogenesis of abnormal pregnancies. Therefore, understanding immune system function during normal pregnancies may provide deeper understanding of immunologically mediated pregnancy complications such as preterm labor and preeclampsia. TLRs are a crucial aspect of the body’s first-line of defense in the innate immune system in identifying foreign antigens and stress-related cell breakdown products with subsequent cytokine release. Initial clinical and basic science studies have implicated the immune system and, specifically TLRs, in normal parturition, preterm labor and pre-eclampsia; yet complete mechanistic understanding remains unknown.

Therefore, we sought to analyze the longitudinal expression of TLRs at standardized collection times prospectively throughout uncomplicated pregnancies and the postpartum state. Initial studies of TLR expression demonstrate a temporal relationship of TLR regulation both on immune cells in the circulation and nonimmune cells in the placenta.1 Innate immune cells, such as dendritic cells, infiltrate the decidua around invading trophoblasts.1 Nonimmune cells such as syncytiotrophoblast cells have also been shown to express TLR 3, 4, 5, 6, and 9 intermittently during pregnancy. Their expression is not constant but rather temporal with certain TLRs on synciotrophoblasts up-regulated only in the third trimester.3,4,18 Therefore, further evaluation of any temporal relationship of posttranslational expression of TLRs in maternal circulation warrants further study.

We chose to evaluate TLR expression in dendritic cells as they are the critical antigen-presenting cells forming the bridge between innate and adaptive immunity; when dendritic cells are depleted, failure of primary adaptive T-cell immunity occurs.19 Dendritic cell activation by specific TLRs results in activation of the adaptive immune system, as they are a key cell type linking innate immunity with T- and B-cell activation.20 Furthermore, unlike placental tissue, circulating dendritic cells may be readily evaluated throughout pregnancy and postpartum allowing for immunologic analysis longitudinally during pregnancy and the puerperium. We analyzed all 9 TLRs hypothesizing that given changes in systemic milieu during the end of pregnancy, there may be systemic changes in other TLRs not previously studied.

Our results describe the longitudinal expression of TLRs 1-9 in maternal dendritic cells in a large cohort of uncomplicated pregnancies from first trimester through the postpartum period. We demonstrate that uncomplicated pregnancies exhibit a proinflammatory milieu on both myeloid and plasmacytoid dendritic cells with longitudinal increases in TLRs 1, 7, and 9 protein levels throughout pregnancy. Post-partum, protein levels of TLR 1 remain elevated compared with nonpregnant levels with TLRs 7 and 9 declining toward levels less than first trimester levels. To our knowledge, these are novel findings and provide important insight into TLR protein expression in the maternal circulation of healthy pregnancies that show a selective activation of TLRs during gestation that primarily resolves postpartum.

Our results are similar to those of Nitsche et al2 who showed that mRNA levels of TLRs 2 and 4 on maternal neutrophils did not significantly change in the maternal circulation longitudinally during uncomplicated pregnancy compared with nonpregnant controls. Results of Nitsche et al2 and ours taken together suggest that TLRs 2 and 4 are not strongly regulated during pregnancy on either neutrophils or dendritic cells. There has been limited evaluation of protein expression of all 9 TLRs in dendritic cells in uncomplicated pregnancies. Studies on TLRs in complicated pregnancies demonstrate systemic TLR dysfunction in preeclampsia6,10 and preterm labor with associated cytokine production of TNFα and IL-6.11–13 Additional research demonstrates elevated trophoblast production of TLR 4 protein in preeclamptic patients compared with patients with preterm labor.21 Because multiple innate and adaptive immune cell types express TLRs, and their expression and function can be differentially regulated, comprehensive TLR evaluation is critical to understanding the immune systems integration during pregnancy.

Our finding that these particular TLRs are elevated above nonpregnant levels is notable given that TLRs 7 and 9 are nucleic acid receptors, which recognize single-stranded RNA and unmethylated CpG DNA. TLR 9 has been identified as a specific receptor for fetal DNA16 and one can postulate that TLR 7 may recognize fetal RNA. Fetal nucleic acids are detectable in maternal plasma beginning in the first trimester and the volume increases as gestation advances; fetal nucleic acids are released into the circulation by a number of processes, notably from cell breakdown of syncio-trophoblasts.22,23 The progressive increase in TLRs 7 and 9 levels may correspond to the innate immune response to increasing amounts of fetal DNA and RNA in maternal circulation. Panda et al10 noted that preeclamptic women had higher levels of circulating basal TLR 9 in plasmacytoid dendritic cells compared with gestational-age matched nonhypertensive pregnant women, hypothesizing that TLR 9 expression is elevated because of the known higher levels of fetal DNA in preeclamptic women. Our data reflects that pregnancy itself and the puerperium without complications results in higher levels of TLR 9 protein compared with nonpregnant controls. Subsequent study correlating fetal DNA levels and immunomodulatory hormones with TLR expression is needed to further explore this theory.

Prior evidence suggests that TLR function may be correlated with exogenous ligands such as DNA/RNA oligonucleotides released from cell-breakdown including cell turnover from stressed placentation.16 A change in the quality or quantity of synciotrophoblast turnover may alter TLR response and increase the risk of preeclampsia or other complications of pregnancy. As dendritic cells are the link to the adaptive immune system and T-cell regulation,19,20 altered regulation of dendritic cells and TLR may lead to altered local and systemic adaptive immune system.

The continued elevation of TLR 1 and IL-12 postpartum above nonpregnant levels is novel and challenges the hypothesis that the immunologic changes experienced during pregnancy resolve by the standard 6-week postpartum visit. These women did not experience any known postpartum complications. We hypothesize that breastfeeding may continue to activate TLR 1 and IL-12 expression by ongoing exposure to low-level gram-positive microorgan. Although there was not a statistical difference in TLR 1 levels between breast-feeding and nonbreastfeeding women (data not shown), the study was not powered to detect this difference as the majority of women in our cohort reported breastfeeding at their postpartum visit. Future investigation on the relationship between TLR 1, IL-12, and breastfeeding may provide insight into aspects of the immunologic state of women during lactation.

Our pregnant and nonpregnant cohorts differed significantly with respect to age and parity. The use of multivariate mixed effect modeling to account for the heterogeneity in our cohort allowed for adjustment for potential confounders although simultaneously using information from all TLR ligand expression. Specifically, we adjusted for body mass index, age, and parity. Given the prospective nature of this study, the mode of delivery for women in the pregnant cohort typically could not be predicted. There is conflicting and limited data based on small studies on particular TLRs evaluating whether labor effects TLR or cytokine expression24–26; and therefore, women were not excluded from analysis based on mode of delivery and the presence of labor was not incorporated into our model as a covariate. The majority of women in our pregnant cohort experienced labor, however, it is possible that our results are impacted by the heterogeneity of delivery mode. In addition, there exists the possibility of unmeasured confounding.

Our study focused on TLR protein expression on dendritic cells to better define the temporal expression of the 9 TLRs in normal pregnancy; our results indicate there are ongoing changes in specific TLR and cytokine expression longitudinally in uncomplicated pregnancies; these findings will allow us to better focus on abnormal expression levels so that we may begin to appreciate the contribution of innate and adaptive immune function in the biology of uncomplicated and pathogenesis of complicated pregnancies.

Acknowledgments

The authors thank Dr Mark Phillippe MD, MHCM of Massachusetts General Hospital for manuscript review.

The above acknowledged individual has no conflict of interest and no funding to report for his contribution.

This research received financial support from Vincent Memorial Research Funds (BRR).

Footnotes

The authors report no conflict of interest.

Presented in poster format at the 33rd annual meeting of the Society for Maternal-Fetal Medicine, San Francisco, CA, Feb. 11–16, 2013.

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