Estradiol alters the immune-responsiveness of cervical epithelial cells stimulated with ligands of Toll-like receptors 2 and 4

Abstract

The mucosa of the female reproductive tract plays a pivotal role in host defence. Pregnancy must alter immunological mechanisms at this interface to protect the conceptus. We sought to determine how estradiol (E2) alters the immune-responsiveness of cervical epithelial cells to ligand stimulation of Toll-like receptor (TLR)-2 and -4. Human ectocervical epithelial cells (HECECs) were cultured and co-incubated with two concentrations of E2 and peptidoglycan (PGN) or lipopolysaccharide (LPS) over durations that ranged between 10 minutes and 18 hours. Cytometric Bead Array was performed to quantify eight cytokines in the supernatant fluid. In response to PGN, HECECs co-incubated with E2 released lesser quantities of IL-1ß and IFNγ, higher levels of RANTES, and variable levels of IL-6 and IL-8 than those not exposed to E2. In contrast, HECECs co-incubated with LPS and E2 secreted increased levels of IL-1ß, IL-6, IL-8, and IFNγ at 2 and 18 hours than HECECs not exposed to E2, and reduced levels of RANTES at same study time-points. Estradiol alters the immune-responsiveness of cultured HECECs to TLR2 and TLR4 ligands in a complex fashion that appears to vary with bacterial ligand, TLR subtype, and duration of exposure. Our observations are consistent with the functional complexity that this mucosal interface requires for its immunological roles.

Fig 1. Cultured HECECs and validating their epithelial nature.

(A & B) Double Immuno-Fluorescence sequential staining for HECECs and HNFF; In “A and B” both antibodies (CK and CD90) and DAPI were used. The detected red signal from the HECECs (A) demonstrates that just the Cytokeratine Ab (PE) was picked up. In “B” detected green signal represents specific Fibroblasts’ antigens identification. The nuclei have been stained with DAPI, validating the cells were alive before fixation. No green signal was detected from the cytoplasm of the culture HECECs (A); indicative of the absence of fibroblasts. HNFF; Human Neonatal Foreskin Fibroblast.

Fig 2. RT-PCR using RNA extracted from Cultured HECECs to investigate TLR2, TLR4, ERs and PRs gene expression.

A: Detection of the signals produced by RT-PCR products for β-Actin, TLR2 and TLR4. B: Depicts detection of ERα. C: Depicts detection of ERβ. D: Signals were detected for mPRα, mPRβ, mPRγ and nPR A&B which represent the gene expression of these five receptors while the detected signals for PRγ are much weaker than the others. No signal was detected in the negative controls, representing the accuracy of the results. Presence of a faint band could be expected in the No-RT controls and it does not interfere with the accuracy of the results.

Fig 3. Flow cytometry results for the study of TLR2 and TLR4 expression in HECECs.

A; TLR2 expression level in HECECs was revealed by detection of fluorochrome signals in histograms; Overlay histograms of isotype control (Red) and an unknown sample (Blue) stained with APC conjugated human TLR2 Ab. The fluorochrome-stained HECECs for TLR2 were highlighted within the interval gate. These defined gates were used to acquire the corresponding statistics. B; TLR4 expression level in the HECECs was revealed by detection of fluorochrome signals in histograms; Overlay histograms of isotype control (Red), unstained HECECs and an unknown sample (Brown) stained with FITC conjugated human TLR4 Ab. The fluorochrome-stained HECECs for TLR4 were highlighted within the interval gate.

Fig 4. Detected cytokine secretion profiles when the cultured HECECs were stimulated with TLR2 agonist.

Demonstrates significant changes in five out of eight studied cytokines when the HECECs were stimulated with PGN in the presence of two different concentrations of E₂ compared to non-E₂-treated. *p value <0.05, **p value<0.005, ***p value<0.0005, ****p value<0.0001.

Fig 5. Detected cytokine secretion profiles when the cultured HECECs were stimulated with TLR4 agonist.

Depicts significant alteration in five out of eight studied cytokine levels when the HECECs were stimulated with LPS in the presence of two different E₂ concentrations compared to non-E₂-treated HECECs. *p value <0.05, **p value<0.005, ***p value<0.0005, ****p value<0.0001.