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

ABSTRACT

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 IC was used as a TLR 3-specific ligand and endometrial cells were either treated or not with Poly IC (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 IC 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 IC 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.