Thymic stromal lymphopoietin participates in the host response to intra-amniotic inflammation leading to preterm labor and birth

Abstract

The aim of this study was to establish the role of thymic stromal lymphopoietin (TSLP) in the intra-amniotic host response of women with spontaneous preterm labor (sPTL) and birth. Amniotic fluid and chorioamniotic membranes (CAM) were collected from women with sPTL who delivered at term (n = 30) or preterm without intra-amniotic inflammation (n = 34), with sterile intra-amniotic inflammation (SIAI, n = 27), or with intra-amniotic infection (IAI, n = 17). Amnion epithelial cells (AEC), Ureaplasma parvum, and Sneathia spp. were also utilized. The expression of TSLP, TSLPR, and IL-7Rα was evaluated in amniotic fluid or CAM by RT-qPCR and/or immunoassays. AEC co-cultured with Ureaplasma parvum or Sneathia spp. were evaluated for TSLP expression by immunofluorescence and/or RT-qPCR. Our data show that TSLP was elevated in amniotic fluid of women with SIAI or IAI and expressed by the CAM. TSLPR and IL-7Rα had detectable gene and protein expression in the CAM; yet, CRLF2 was specifically elevated with IAI. While TSLP localized to all layers of the CAM and increased with SIAI or IAI, TSLPR and IL-7Rα were minimal and became most apparent with IAI. Co-culture experiments indicated that Ureaplasma parvum and Sneathia spp. differentially upregulated TSLP expression in AEC. Together, these findings indicate that TSLP is a central component of the intra-amniotic host response during sPTL.

Keywords: Amniotic fluid; Chorioamniotic membranes; Sneathia; TSLP; Ureaplasma.

Figure 1.. Concentrations of TSLP in amniotic fluid of women with spontaneous preterm labor.

(A) Representative diagram showing the study groups of women who underwent spontaneous preterm labor and delivered at term (PTL del Term) and those who delivered preterm without intra-amniotic inflammation (PTL-No SIAI/IAI), with sterile intra-amniotic inflammation (PTL-SIAI), or with intra-amniotic infection (PTL-IAI) from whom amniotic fluid was collected to measure TSLP. (B) TSLP concentrations in the amniotic fluid of women with PTL del Term (n = 30), PTL No SIAI/IAI (n = 34), PTL SIAI (n = 27), and PTL IAI (n = 17). Data are shown as box and whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum/maximum values. P-values were determined using the Kruskal-Wallis test followed by Dunn’s post-hoc test. ***p < 0.001, ****p < 0.0001.

Figure 2.. The expression of TSLP and its receptors in the chorioamniotic membranes.

(A) Representative diagram showed the detection of TSLP and its receptors (TSLPR and IL-7Rα) in the chorioamniotic membranes (CAM). (B) RT-qPCR assay showing the gene expression of TSLP in the CAM of women with PTL del Term, PTL No SIAI/IAI, PTL SIAI, and PTL IAI (n = 6 per group). (C) Representative western blot image and relative quantification showing the protein expression of TSLP (10 kDa) in the CAM (n = 3 per group). (D) Representative immunofluorescence staining showing the expression of TSLP (red signal) by amniotic fluid neutrophils. White arrows indicate positive TSLP staining. DAPI staining (blue) shows cell nuclei. The isotype (negative control) staining images in each group are shown at the left corner. All images taken at 400X magnification. Scale bar represents 5 μm. RT-qPCR assays showing the gene expression of (E) CRLF2 and (G) IL7R in the CAM (n = 6 per group). Representative western blot images and relative quantification showing the protein expression of (F) TSLPR (53 kDa) and (H) IL-7Rα (51 kDa) in the CAM (n = 3 per group). RT-qPCR data are shown as box and whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum/maximum values. β-actin (ACTB) (42 kDa) was used as internal control for western blot analysis. P-values were determined by Mann-Whitney U-tests. *p < 0.05.

Figure 3.. Spatial expression of TSLP and its receptors in the chorioamniotic membranes.

(A) Representative immunohistochemistry images showing the expression of TSLP, TSLPR, and IL-7Rα (brown staining) in the amnion, chorion, or decidua of the CAM from women with PTL del Term, PTL No SIAI/IAI, PTL SIAI, and PTL IAI (n = 5 per group). All images were taken at 200X digital magnification. Scale bar represents 50 μm. Tissue layers are indicated as A: Amnion; C: Chorion; D: Decidua. (B) Dot plots representing the expression of TSLP, CRLF2, and IL7R in the CAM and amnion of women with PTL del Term, PTL No SIAI/IAI, PTL SIAI, and PTL IAI (n = 6 per group). Dotted horizontal lines correspond to 40 cycles, which was considered as the detection limit threshold. Blue dots represent CAM, red dots represent amnion.

Figure 4.. Ureaplasma parvum stimulates TSLP expression in human amnion epithelial cells.

(A) Representative diagram showing the co-culture of human amnion epithelial cells (AEC) (n = 3 samples with 3 technical replicates each) with two isolates of U. parvum (from a woman with term or preterm labor) or vehicle control (SP4 broth) for 24 h, after which AEC were collected to determine TSLP expression. (B) TSLP expression in AEC incubated with SP4 borth or U. parvum isolates. Data are shown as box and whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum/maximum values. P-values were determined by Mann-Whitney U-tests. ****p <0 .0001.

Figure 5.. Sneathia spp. stimulate TSLP expression in amnion epithelial cells.

(A) Representative diagram the co-culture of human amnion epithelial cells (AEC) (n = 3 samples with 3 technical replicates each) with Sneathia vaginalis (S.V.), Sneathia sanguinegens (S.S.), or vehicle control [supplemented (s)BHI broth, see Methods] for 24 h, after which the AEC were collected to determine TSLP expression. (B) TSLP expression in AEC incubated with sBHI broth, S.V., or S.S. Data are shown as box and whisker plots where midlines indicate medians, boxes indicate interquartile ranges, and whiskers indicate minimum/maximum values. P-values were determined by Mann-Whitney U-tests. *p < 0.05, ***p < 0.001. (C) Representative immunofluorescence images showing the expression of TSLP (red signal) in AEC treated with sBHI broth, S.V., or S.S. White arrows indicate positive TSLP staining. DAPI staining (blue signal) shows cell nuclei. The isotype (negative control) staining images in each group are shown at the left corner. All images taken at 400X magnification. Scale bar represents 20 μm.