Interleukin-1 receptor antagonist mediates toll-like receptor 3-induced inhibition of trophoblast adhesion to endometrial cells in vitro.

STUDY QUESTION: Is interleukin-1 receptor antagonist (IL-1RA) involved in the toll-like receptor 3 (TLR 3)-induced inhibition of trophoblast cells' adhesion to endometrial cells in vitro? SUMMARY ANSWER: IL-1RA mediates the TLR 3-induced inhibition of trophoblast cells' adhesion to endometrial cells in vitro. WHAT IS KNOWN ALREADY: It is well documented that endometrial TLR 3 activation leads to impairment of trophoblast binding to endometrial cells in vitro. IL-1RA is known as an anti-implantation factor, as its injection significantly reduced implantation rates in mice by an effect on endometrial receptivity. STUDY DESIGN, SIZE, DURATION: Poly I:C was used as a TLR3 specific ligand and endometrial cells were either treated or not with Poly I:C (treated versus control) in vitro. IL-1RA was applied to block IL-1 signal transduction. IL-1RA was knocked down by Accell Human IL1RN siRNA. Flagellin was used to stimulate TLR 5. SP600125 (JNK) was applied to inhibit the mitogen-activated protein kinases (MAPK) pathway. BAY11 -7082 was used to inhibit the nuclear factor-κB (NF-κB) pathway. The experiments were performed in three replicates on three separate days. PARTICIPANTS/MATERIALS, SETTING, METHODS: An in vitro assay was developed using RL95-2 (an endometrial cell line) and JAr (a trophoblast cell line) cells. Initially, the production of IL-1RA in RL95-2 cells in response to TLR 3 activation was measured. To determine whether the TLR 3-induced inhibition of trophoblast binding was mediated through IL-1RA: (i) we evaluated the effect of IL-1RA on the attachment of trophoblast cells to endometrial cells; (ii) we knocked down TLR3-induced IL-1RA gene expression by IL-1RA Small interfering RNA (siRNA) and evaluated trophoblast attachment to endometrial cells. Finally, to clarify through which pathway TLR 3-induced inhibition of trophoblast binding occurs: (i) activation of NF-κB and MAPK was detected by transfecting the cells with secreted placental alkaline phosphatase reporter plasmids bearing promoter sequences for each transcription factor; (ii) the inhibitors for NF-κB and MAPK were used to block signaling; (iii) it was then investigated whether addition of these inhibitors could restore the TLR 3-induced impairment of trophoblast attachment to the endometrial cells. MAIN RESULTS AND THE ROLE OF CHANCE: Our results showed that addition of polyinosinic:polycytidylic acid (Poly I:C) to RL95-2 cells significantly increased the production of IL-1RA (P < 0.05). Addition of human recombinant IL-1RA to RL95-2 cells remarkably decreased the adhesion rate of trophoblast cells to endometrial cells (P < 0.05). In addition, suppression of TLR3-induced IL-1RA gene expression in RL95-2 cells significantly restored trophoblast cells attachment to endometrial cells in the presence of Poly I:C (P < 0.05). Only TLR3 and not TLR5 induced MAPK activation (P < 0.05). TLR3 ligation did not affect NF-κB activation. Of NF-kB and MAPK inhibitors, only MAPK's inhibitor could achieve restoration of spheroid adhesion to endometrial cells (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This study has been only done in vitro. Future in vivo studies will confirm our data. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study have a potential clinical application in introducing IL-1RA as one of the diagnostic infertility markers in the endometrium, which can affect the process of embryo adhesion at the time of implantation. Moreover, based on the novel data obtained in the current study, blocking and regulating the MAPK pathway by its inhibitors can be used as a new strategy to prevent and treat virus-induced infertility cases in ART techniques. STUDY FUNDING/COMPETING INTEREST: This study was partially funded by a Marie Curie IIF-253948 grant to I.C. and was partially funded by the author's institutions. The authors have no conflict of interest to declare.

Activation of Toll-like receptor 3 reduces actin polymerization and adhesion molecule expression in endometrial cells, a potential mechanism for viral-induced implantation failure.

STUDY QUESTION: Does activation of endometrial Toll-like receptor 3 (TLR 3) affect cell receptivity to trophoblast adhesion? SUMMARY ANSWER: TLR 3 activation in vitro reduces the attachment of trophoblast cells to endometrial cells by altering the cell cytoskeleton and reducing the expression of adhesion molecules in human endometrial cells. WHAT IS KNOWN ALREADY: It is well documented that the presence of an infection at the time of implantation can lead to implantation failure. The female reproductive tract recognizes invading micro-organisms through the innate pathogen recognition receptors such as the TLRs. STUDY DESIGN, SIZE, DURATION: Poly I:C was used as a TLR 3-specific ligand and endometrial cells were either treated or not with Poly I:C (treated versus control) in vitro. The experiments were performed in three replicates on three separate days. PARTICIPANTS/MATERIALS, SETTING, METHODS: An in vitro assay was developed using RL95-2 (a human endometrial cell line) and JAr (a human trophoblast cell line) cells. Initially, the percentage of attached JAr spheroids to RL95-2 was measured in response to TLR 3 activation. Next, actin polymerization in RL95-2 cells was assessed in response to TLR 2/6, 3 and 5 activation. Phalloidin was used to assess the mean fluorescence intensity of F-actin by flow cytometry or confocal microscopy. Secondly, the influence of TLR 2/6, 3 and 5 activation on the expression of cluster of differentiation 98 (CD98) and ß3 integrin was determined. To further understand through which pathways the TLR 3-induced alterations occur, inhibitors were applied for Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-beta (TRIF), myeloid differentiation primary response 88 (MYD88), mitogen-activated protein kinases (MAPK) and nuclear factor pathways. MAIN RESULTS AND THE ROLE OF CHANCE: We observed that stimulation of TLR 3 in endometrial cells with different concentrations of Poly I:C led to a reduction in the percentage of trophoblasts attached to the endometrial cells in a dose-dependent manner (P < 0.05). This decrease was consistent in the Poly I:C treated group regardless of the co-incubation time (P < 0.05). In addition, our results demonstrated that actin polymerization and CD98 expression significantly decreased only in response to TLR 3 activation (P < 0.05). Activation of endometrial cells with TLR 2/6, 3 and 5 significantly reduced ß3 integrin expression (P < 0.05). These alterations were shown to work via MYD88-MAPK pathways (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This study has been performed in vitro. Future in vivo studies will be required in order to confirm our data. WIDER IMPLICATIONS OF THE FINDINGS: This is a novel discovery which extends our current knowledge concerning diagnosis and treatment of viral-induced infertility cases. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the COST Action FA1201 (GEMINI) by granting a Short Term Scientific Mission and the Instituto de Salud Carlos III by granting Grant PI11/01645. The authors have no conflict of interest to declare.