The Th1:Th2 Dichotomy of Pregnancy and Preterm Labour

Pregnancy is a unique immunological state in which a balance of immune tolerance and suppression is needed to protect the fetus without compromising the mother. It has long been established that a bias from the T helper 1 cytokine profile towards the T helper 2 profile contributes towards successful pregnancy maintenance. The majority of publications that report on aberrant Th1:Th2 balance focus on early pregnancy loss and preeclampsia. Over the last few decades, there has been an increased awareness of the role of infection and inflammation in preterm labour, and the search for new biomarkers to predict preterm labour continues. In this paper, we explore the evidence for an aberrant Th1:Th2 profile associated with preterm labour. We also consider the potential for its use in screening women at high risk of preterm labour and for prophylactic therapeutic measures for the prevention of preterm labour and associated neonatal adverse outcomes

Changes in the Th1 : Th2 Cytokine Bias in Pregnancy and the Effects of the Anti-Inflammatory Cyclopentenone Prostaglandin 15-Deoxy-Δ12,14-Prostaglandin J2

Pregnancy is a complex immunological state in which a bias towards T helper 2 (Th2) protects the fetus. Evidence suggests that proinflammatory cytokines increase the risk of poor neonatal outcome, independently of the direct effect of preterm labour. The anti-inflammatory prostaglandin 15-deoxy-Δ12,14-Prostaglandin J2 (15dPGJ2) inhibits nuclear factor Kappa B (NF-κB) in amniocytes and myocytes in vitro and is a ligand for the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) receptor. Here we examine the Th1:Th2 cytokine bias in pregnancy and whether 15dPGJ2 could be used to inhibit the production of the proinflammatory cytokines through inhibition of NF-κB while simultaneously promoting Th2 interleukin 4 (IL-4) synthesis via CRTH2 in T helper cells. Peripheral blood mononuclear cells (PBMCs) from women at 28 weeks, term pre-labour, term labour as well as non-pregnant female controls were cultured with 15dPGJ2 or vehicle control and stimulated with phorbol myristyl acetate (PMA)/ionomycin. The percentage of CD4+ cells producing interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in response to PMA/ionomycin was significantly reduced in pregnancy. 15dPGJ2 reduced IFN-γ and TNF-α production in stimulated T helper cells, but did not alter IL-4 production in CRTH2+ve cells. 15dPGJ2 also reduced phospho-p65 in stimulated PBMCs. In summary, 15dPGJ2 suppresses the Th1 response of PBMCs during pregnancy and active labour whilst maintaining the Th2 response suggesting a therapeutic benefit in reducing neonatal morbidity in inflammation-induced PTL

Chemoattractant Receptor Homologous to the T Helper 2 Cell (CRTH2) Is Not Expressed in Human Amniocytes and Myocytes

BACKGROUND: 15-deoxy-Δ 12,14- Prostaglandin J2 (15dPGJ2) inhibits Nuclear factor kappa B (NF-κB) in human myocytes and amniocytes and delays inflammation induced preterm labour in the mouse. 15dPGJ2 is a ligand for the Chemoattractant Receptor Homologous to the T helper 2 cell (CRTH2), a G protein-coupled receptor, present on a subset of T helper 2 (Th2) cells, eosinophils and basophils. It is the second receptor for Prostaglandin D2, whose activation leads to chemotaxis and the production of Th2-type interleukins. The cellular distribution of CRTH2 in non-immune cells has not been extensively researched, and its identification at the protein level has been limited by the lack of specific antibodies. In this study we explored the possibility that CRTH2 plays a role in 15dPGJ2-mediated inhibition of NF-κB and would therefore represent a novel small molecule therapeutic target for the prevention of inflammation induced preterm labour. METHODS: The effect of a small molecule CRTH2 agonist on NF-κB activity in human cultured amniocytes and myocytes was assessed by detection of p65 and phospho-p65 by immunoblot. Endogenous CRTH2 expression in amniocytes, myocytes and peripheral blood mononuclear cells (PBMCs) was examined by PCR, western analysis and flow cytometry, with amniocytes and myocytes transfected with CRTH2 acting as a positive control in flow cytometry studies. RESULTS: The CRTH2 agonist had no effect on NF-κB activity in amniocytes and myocytes. Although CRTH2 mRNA was detected in amniocytes and myocytes, CRTH2 was not detectable at the protein level, as demonstrated by western analysis and flow cytometry. 15dPGJ2 inhibited phospho-65 in PBMC'S, however the CRTH2 antagonist was not able to attenuate this effect. In conclusion, CRTH2 is not expressed on human amniocytes or myocytes and plays no role in the mechanism of 15dPGJ2-mediated inhibition of NF-κB

Characterization of neutrophil subsets in healthy human pregnancies.

We have previously shown that in successful pregnancies increased arginase activity is a mechanism that contributes to the suppression of the maternal immune system. We identified the main type of arginase-expressing cells as a population of activated low-density granulocytes (LDGs) in peripheral blood mononuclear cells and in term placentae. In the present study, we analyzed the phenotype of LDGs and compared it to the phenotype of normal density granulocytes (NDGs) in maternal peripheral blood, placental biopsies and cord blood. Our data reveal that only LDGs but no NDGs could be detected in placental biopsies. Phenotypically, NDGs and LDGs from both maternal and cord blood expressed different levels of maturation, activation and degranulation markers. NDGs from the maternal and cord blood were phenotypically similar, while maternal, cord and placental LDGs showed different expression levels of CD66b. LDGs present in cord blood expressed higher levels of arginase compared to maternal and placental LDGs. In summary, our results show that in maternal and cord blood, two phenotypically different populations of neutrophils can be identified, whereas in term placentae, only activated neutrophils are present

First trimester circulating MicroRNA biomarkers predictive of subsequent preterm delivery and cervical shortening

Preterm birth (PTB) is the leading cause of infant death and disability worldwide. The onset of preterm uterine contractions is preceded by asymptomatic cervical remodelling and ripening, which can be seen on trans-vaginal ultrasound as cervical shortening. This study aimed to identify plasma miRNA biomarkers that predict preterm birth and/or cervical shortening. We collected serial plasma samples from pregnant women prospectively from 12 to 22 weeks gestation. The nCounter miRNA assay was used to identify differentially expressed miRNAs associated with spontaneous PTB and/or cervical shortening (n = 16 term no short, n = 13 preterm, n = 24 short). Predictive values of the miRNA biomarkers were confirmed in an independent validation cohort consisting of 96 women who delivered at term, 14 preterm and 21 early cervical shortening at <20 weeks gestation. Nine miRNAs (hsa-let-7a-5p, hsa-miR-374a-5p, hsa-miR-15b-5p, hsa-miR-19b-3p, hsa-miR-23a-3p, hsa-miR-93-5p, hsa-miR-150-5p, hsa-miR-185-5p and hsa-miR-191-5p) were differentially expressed (P < 0.001) in women subsequently experiencing PTB or cervical shortening. Hsa-miR-150-5p had the strongest ability to predict PTB (AUC = 0.8725) and cervical shortening (AUC = 0.8514). Plasma miRNAs in the first trimester can predict PTB and cervical shortening in women at risk of preterm delivery. This is a key period in pregnancy when early identification of PTB risk allows time to deliver outcome-modifying interventions

Flow cytometry for the detection of endogenous CRTH2.

<p>Lymphocytes were gated according to forward scatter and side scatter and T helper cells were identified using CD4 as a cell surface marker (n = 6). A representative cytogram is presented with the right upper quadrant showing CRTH2⁺/CD4⁺ lymphocytes (<b>A</b>). Histograms showing no staining, isotype control and CRTH2⁺ labelled lymphocytes (<b>B</b>), amniocytes (<b>C</b>) and myocytes (<b>D</b>), (n = 5).</p

15dPGJ2 reduced NF-κB p65 activity in amniocytes and myocytes.

<p>Protein was extracted from IL-1β stimulated and 15dPGJ2 treated cells and levels of nuclear p65 were examined using immunoblotting. A dose response of 0.1–32 µM of 15dPGJ2 was used (n = 3). Representative immunoblots are shown for amniocytes, (<b>A</b>), myocytes (<b>B</b>). Immunoblots were re-probed for β-actin as an internal loading control.</p

Western blot of protein lysate from PBMCs, amniocytes, myocytes.

<p>50 µg of whole cell lysate from PBMCs, amniocytes, myocytes were subject to western analysis. Membranes were probed with the primary antibody -SC-23092 to detect a Mr∼34 000 product (<b>A</b>; Lanes 1–3). The secondary antibody control blot is also shown (<b>B</b>; Lanes 1–3). β-actin was used as a loading control. Lane 1 = PBMCs, lane 2 = amniocytes, lane 3 = myocytes. Multiple bands are seen on the blot. A band appears at Mr∼34 000 in amniocytes, faintly in the myocyte lane, but is absent in the positive control PBMCs lane. However, the strongest bands appear at Mr∼15 000 and at just above Mr∼43 000 in all lanes. Myocytes from passage zero, one, two, three and four (lanes 1–5 respectively) were also examined for CRTH2 expression showing a very faint band at Mr∼34 000, with no effect of passage number (<b>C</b>).</p

Pyl A has no effect on NF-κB p65 activity in amniocytes and myocytes.

<p>Protein was extracted from IL-1β stimulated and Pyl treated cells and levels of nuclear p65 and phosphorylated p65 (p-p65) were examined using immunoblotting. A dose response of 0.1–32 µM of Pyl A was used. Representative immunoblots are shown for amniocytes, (<b>A</b>) and myocytes (<b>B</b>). Immunoblots were re-probed for β-actin as an internal loading control. Densitometric analysis of the immunoblots was conducted revealing no effect of Pyl A on p65 or p-p65 in amniocytes (<b>C</b>, <b>E</b>) or myocytes (<b>D</b>, <b>F</b>). NS = non-stimulated (non-IL-1β treated cells). Effect of treatment was examined for statistical significance using ANOVA of repeated measures with Bonferroni’s multiple comparison test; *<i>P</i><0.05.</p