Inflammatory Signalling in Fetal Membranes: Increased Expression Levels of TLR 1 in the Presence of Preterm Histological Chorioamnionitis

Abstract

Histological chorioamnionitis (HCA) is an established marker of ascending infection, a major cause of preterm birth. No studies have characterised the global change in expression of genes involved in the toll-like receptor (TLR) signalling pathways in the presence of HCA in the setting of preterm birth (pHCA). Fetal membranes were collected immediately after delivery and underwent histological staging for inflammation to derive 3 groups; term spontaneous labour without HCA (n = 9), preterm birth <34 weeks gestation without HCA (n = 8) and pHCA <34 weeks (n = 12). Profiling arrays ran in triplicate for each group were used to determine the expression of 84 genes associated with TLR signalling and screen for genes of interest (fold change >2; p<0.1). Expression of identified genes was validated individually for all samples, relative to GAPDH, using RT-PCR. Expression of TLR 1, TLR 2, lymphocyte antigen 96, interleukin 8 and Interleukin-1 receptor-associated kinase-like 2 was increased in pHCA (p<0.05). Degree of expression was positively associated with histological staging of both maternal and fetal inflammation (p<0.05). The inflammatory expression profile at the maternal/fetal interface associated with pHCA, a reflection of ascending infection, is extremely heterogeneous suggesting polymicrobial involvement with activation of a common pathway. Antagonism of TLR 1 and TLR 2 signalling in this setting warrants further assessment.

Fig 1. Screening for genes interest: Analysis of TLR pathway signalling array.

Volcano plots were used to identify genes showing significant expression. The example shown here is a volcano plot of PTL^+CA vs TSL^-CA in the amnion (2a). The plot shows the values for all genes in the qPCR array plotted against fold changes. On the Y-axis, values higher than the blue line threshold represent significant genes with an adjusted probability value of <0.1. On the X-axis, values outside the grey lines represent a fold change of either ≥2 or ≤-2. The clustergram with heat map (2b) shows all 84 genes in qPCR array for each group in the amnion, this was used to visually identify the pattern of which clusters of genes showed higher expression in each group.

Fig 2. Expression of TLR 1 is negatively related to gestational age.

Least squares linear regression is used to assess correlation between expression of TLR 1 and gestational age in the absence of chorioamnionitis (PTL^-CA and TSL^-CA). All individual samples with histological confirmation of the absence of CA are shown. TLR 1 shows a strong correlation between decreasing gestational age and increased expression (R² = 0.671, p<0.001). The correlation is also present in the chorion (R² = 0.411, p = 0.006). Expression normalised to GapDH. Gene expression assessed by fold change (2^ΔΔCT).

Fig 3. Fig 3a TLR expression in the amnion: Preterm labour with and without chorioamnionitis is associated with increased expression of TLR 1 and TLR 2 compared to term labour.

All individual samples represented on plot, black line represents mean of samples. A significantly increased mean expression of TLR1and TLR 2 was noted in both the preterm birth groups when comparing to TSL^-CA. (TLR1; PTL^+CA mean expression 82.58 vs TSL^-CA; p = 0.035, PTL^-CA mean expression 32.73 vs TSL^-CA; p = 0.002) (TLR2; PTL^+CA mean expression 80.33 vs TSL^-CA; p = 0.046, PTL^-CA mean expression 12.04 vs TSL^-CA; p = 0.020). Fig 3b TLR expression in the chorion: Histological chorioamnionitis is associated with increased expression of TLR 1 and TLR 2. All individual samples represented on plot, black line represents mean of samples. In the chorion the PTL^+CA group showed a significantly increased expression of TLR1 and TLR2. (TLR1; PTL^+CA mean expression 18.81 vs TSL^-CA; p = 0.001, PTL^+CA mean expression 6.93 vs PTL^-CA; p = 0.040) (TLR2; PTL^+CA mean expression 9.35 vs TSL^-CA; p<0.001, PTL^+CA mean expression 6.93 vs PTL^-CA; p = 0.008). Expression normalised to GapDH. Gene expression assessed by fold change (2^ΔΔCT).

Fig 4. Severity of histological staging of chorioamnionitis correlates with expression of TLR 1 and TLR 2.

This figure shows the relationship between expression of TLR 1 and TLR 2 in the chorion and staging of maternal (4a) and fetal (4b) inflammation. Least squares linear regression was used. All samples staged via Redline criteria included. Higher staging of maternal inflammation correlates with higher expression of TLR1 (R² = 0.332; p = 0.008) and TLR 2 (R² = 0.475; p = 0.001). Higher staging of fetal inflammation shows a similar pattern for TLR 1 (R² = 0.199; p = 0.049) and TLR 2 (R² = 0.433; p = 0.002). Expression normalised to GapDH. Gene expression assessed by fold change (2^ΔΔCT).