IL-1 and TNF mediates IL-6 signaling at the maternal-fetal interface during intrauterine inflammation

Abstract

Introduction: IL6 signaling plays an important role in triggering labor and IL6 is an established biomarker of intrauterine infection/inflammation (IUI) driven preterm labor (PTL). The biology of IL6 during IUI at the maternal-fetal interface was investigated in samples from human subjects and non-human primates (NHP).

Methods: Pregnant women with histologic chorioamnionitis diagnosed by placenta histology were recruited (n=28 term, n=43 for preterm pregnancies from 26-36 completed weeks of gestation). IUI was induced in Rhesus macaque by intraamniotic injection of lipopolysachharide (LPS, n=23). IL1 signaling was blocked using Anakinra (human IL-1 receptor antagonist, n=13), and Tumor necrosis factor (TNF) signaling was blocked by anti TNF-antibody (Adalimumab n=14). The blockers were given before LPS. All animals including controls (intraamniotic injection of saline n=27), were delivered 16h after LPS/saline exposure at about 80% gestation.

Results: IUI induced a robust expression of IL6 mRNAs in the fetal membranes (chorion-amnion-decidua tissue) both in humans (term and preterm) and NHP. The major sources of IL6 mRNA expression were the amnion mesenchymal cells (AMC) and decidua stroma cells. Additionally, during IUI in the NHP, ADAM17 (a protease that cleaves membrane bound IL6 receptor (IL6R) to release a soluble form) and IL6R mRNA increased in the fetal membranes, and the ratio of IL6 and soluble forms of IL6R, gp130 increased in the amniotic fluid signifying upregulation of IL6 trans-signaling. Both IL1 and TNF blockade suppressed LPS-induced IL6 mRNAs in the AMC and variably decreased elements of IL6 trans-signaling.

Discussion: These data suggest that IL1 and TNF blockers may be useful anti-inflammatory agents via suppression of IL6 signaling at the maternal-fetal interface.

Keywords: amnion; chorioamnionitis; inflammation; innate immunity; reproductive immunology.

Figure 1

Chorioamnionitis increased the expression of IL6 mRNA in human term and preterm fetal membranes. Human extraplacental chorioamnion-decidua (CAMD, fetal membranes) samples from women that delivered at term or preterm were obtained within 30 minutes of delivery and chorioamnionitis was diagnosed by placenta histology. (A) Chorioamnionitis (chorio) induces a significant increase of IL6 mRNA expression in both preterm and term samples. (B) Chorio induces a significant decrease of IL6ST (gp130) in term CAMD samples compared to term without chorio (Term chorio neg) samples. (Term chorio neg n=18; Term chorio pos n=9–10; Preterm chorio neg n=18–21; Preterm chorio pos n=15–25). Expression of gene mRNA was measured by quantitative PCR (Taqman probes) and average mRNA values are fold increases over the average value for no chorio after internally normalizing to the housekeeping 18S RNA. Data are mean ± SEM, *p < 0.05 vs. samples without chorio (Mann–Whitney U-test).

Figure 2

Chorio increased significantly the expression of IL6 mRNA in each of the three tissues (amnion, chorion, decidua) of term samples and in the chorion of preterm samples. Amnion, chorion, and decidua parietalis were physically separated. (A) Chorio induces a significant increase of IL6 mRNA expression in the three layers of term samples (Term neg n=15; Term pos n=10). (B) Similar results were observed in the chorion of preterm samples, (Preterm chorio neg n=18–19; Preterm chorio pos n=12–13). Data are mean ± SEM, *p < 0.05 vs. samples without chorio (Mann–Whitney U-test).

Figure 3

Chorio significantly increased the number of human IL6 ⁺ stroma cells in the decidua parietalis and IL6 ⁺ mesenchymal cells in the amnion. Fixed fetal membranes (chorion-amnion-decidua parietalis) paraffin embedded sections were stained by immuno-colocalization. (A) Representative images showing IL6 mRNA identified by RNAscope in situ hybridization and Vimentin (Vim) colocalization by immunofluorescence. IL6 is shown in red and Vim in green. White arrows in the magnified yellow inset (#1-term, #2-preterm) indicate Vim⁺ IL6 ⁺ Amniotic Mesenchymal Cells (AMCs), while white arrows in the magnified orange inset (#3-term, #4-preterm) indicate Vim⁺ IL6 ⁺ Decidual Stromal Cells (DSCs). (B) Representative images showing IL6 mRNA identified by RNAscope in situ hybridization and Pan-cytokeratin (Pancytok) colocalization by immunofluorescence. IL6 is shown in red and Pancytok in green. DAPI indicates nuclear staining (blue) in all images. White arrows in the magnified blue inset indicate Pancytok⁺ IL6 ⁺ Extra Villous Trophoblast (EVTs) in both Term pos (inset #5) and Preterm pos (inset #6) subjects. (a, amnion; c, chorion, and d, decidua) (C) Quantification of Vim⁺ DSC, AMC, and Pancytok⁺ EVT cells expressing IL6 in amnion, chorion, and decidua. Average of 5 randomly selected HPF fields were plotted as the representative value for the sample. Counts were performed in a blinded manner. Data are mean ± SEM. HPF, high-power field; (Term neg n=5; Term pos n=4), (Preterm chorio neg n=4; Preterm chorio pos n=5). *p < 0.05 (Mann–Whitney U-test).

Figure 4

Chorioamnionitis increased the expression of IL6 mRNA in rhesus preterm fetal membranes in a IL1- and TNF-dependent fashion. Chorioamnionitis was induced by intraamniotic (IA) injection of LPS. Controls (Ctrl) received intraamniotic saline. Rhesus extraplacental chorioamnion-decidua (fetal membranes) samples were obtained at delivery 16h after IA LPS/saline and chorioamnionitis (chorio) was diagnosed by placenta histology. mRNAs for molecules in IL6 signaling were measured. (A) Chorio induces a significant increase of IL6 mRNA expression. Both inhibitors Anakinra (Anak) and Adalimumab (Adal) significantly decreased IL6 mRNA expression compared to LPS-exposed animals. (Ctrl n=27; LPS n=23; Anak+LPS n=13; Adal+LPS n=14). (B) Chorio induces a significant increase of ADAM17, IL6R, and STAT3 mRNAs in LPS-exposed samples compared to ctrl samples. Anakinra but not Adalimumab decreased LPS-induced ADAM17 mRNA. (Ctrl n=18; LPS n=16; Anak+LPS n=12; Adal+LPS n=12). Average mRNA values are fold increases over the average value for no chorio (dotted line) after internally normalizing to the housekeeping 18S RNA. Data are mean ± SEM, *p < 0.05 (Mann–Whitney U-test).

Figure 5

Partial decrease of LPS-induced IL6 mRNAs in fetal membranes by Anakinra and Adalimumab. Amnion, chorion, and decidua parietalis were physically separated. LPS-exposure induced a significant increase of IL6 mRNA expression in the three tissue layers. Anakinra decreased LPS-induced IL6 mRNA expression in amnion, while Adalimumab decreased its expression in both amnion and chorion. The inhibitors did not have efficacy in decidua parietalis. Data are mean ± SEM, *p < 0.05 (Mann–Whitney U-test).

Figure 6

LPS-exposure significantly increased IL6/sIL6R and IL6/sgp130 ratios in the amniotic fluid. Cytokine concentrations were measured in amniotic fluid by ELISA. (A) Soluble IL6R (sIL6R) and (B) soluble gp130 (sgp130) protein levels did not change after LPS exposure. (C) Graph shows a significant increase in IL6/sIL6R and IL6/sgp130 ratio after LPS-exposure. Data are mean ± SEM, *p < 0.05 (Mann–Whitney U-test) (Ctrl n=6; LPS n=6; Anak + LPS n=6; Adal + LPS n=6).

Figure 7

LPS exposure increased significantly the number of rhesus IL6 ⁺ stroma cells in the decidua parietalis and IL6 ⁺ amnion mesenchymal cells. Fixed fetal membranes (chorion-amnion-decidua parietalis) paraffin embedded sections were stained by immuno-colocalization. (A) Representative images showing IL6 mRNA identified by RNAscope in situ hybridization and Vim colocalization by immunofluorescence. IL6 is shown in red and Vim in green. White arrows in the magnified yellow inset 1 indicate Vim⁺ IL6 ⁺ Amnion mesenchymal cells (AMCs), while white arrows in the magnified orange inset 2 indicate Vim⁺IL6⁺ Decidua stroma cells (DSCs) in LPS-exposed animals. (B) Quantification of Vim⁺ DCSs and Vim⁺ AMCs expressing IL6 in amnion, chorion, and decidua. (C) Representative images showing IL6 mRNA identified by RNAscope in situ hybridization and Pancytok colocalization by immunofluorescence. IL6 is shown in red and Pancytok in green. White arrows in the magnified blue inset #3 indicate Pancytok⁺ IL6 ⁺ EVTs in CAMD section of Anak+LPS animal. subjects. DAPI indicates nuclear staining (blue) in all images. (D) Quantification of Pancytok⁺ extra villous trophoblast (EVT) cells expressing IL6 in amnion, chorion, and decidua. For quantification, an average of 5 randomly selected HPF fields were plotted as the representative value for the animal. Counts were performed in a blinded manner. HPF, high-power field. Data are mean ± SEM, *p < 0.05 (Mann–Whitney U-test) (Ctrl n=4; LPS n=5; Anak+LPS n=5; Adal+LPS n=5). HPF, high-power field; a, amnion; c, chorion, and d, decidua.

Figure 8

Model for IL6 signaling at the maternal-fetal interface during IUI. A schematic model based on data from Rhesus macaque and human subjects showing source and signaling of IL6 in normal pregnancy and during IUI. (A) Under normal conditions, a small amount of IL6 is expressed in the amnion and decidua. IUI significantly increases IL6 expression in activated amnion mesenchymal cells (AMC), decidua stroma cells, and to a small extent in chorion trophoblast cells (not shown). IL6 expression by AMC is inhibited by IL1- and TNF-inhibitors. (B) During normal pregnancy IL6 signaling occurs in restricted cells expressing IL6 receptor (IL6R) and gp130 and serves homeostatic function (note both IL6R and gp130 are cell membrane spanning receptors). Soluble IL6R (sIL6R) and soluble gp130 (sgp130) lacking membrane spanning domains are secreted in normal conditions. Due to low levels of IL6 in normal conditions, no significant IL6-sIL6R interactions occur. During IUI, large increase of IL6 expression occurs at the maternal-fetal interface, which is inhibited by IL1- and TNF- inhibitors. Increased IL6 concentration promotes increased number of cells signaling via classical IL6-IL6R-gp130 signaling. sIL6R secretion increases slightly during IUI and sgp130 secretion tends to decrease slightly. Increased IL6 in the inflammatory mileu increases binding to sIL6R. This complex can now bind to membrane spanning gp130 that is expressed ubiquitously. This IL6 trans-signaling increases the number and types of cells responding to IL6 and is pro-inflammatory in nature.