Platelet secretions exert anti-inflammatory effects <i>in vitro</i> on neutrophils and uterine epithelia in cattle: a possible role in amplifying the uterine immune network toward pregnancy.

Abstract

<h4>Background</h4>Platelet derivatives improve the uterine immune environment and increase pregnancy success in humans and animals. Platelet-conditioned media (PCM) contain all molecules derived from platelets <i>in vitro</i> (platelet secretions). The present study aimed to investigate the immunological impacts of platelet secretions on polymorphonuclear neutrophils (PMNs) and bovine endometrial epithelial cells (BEECs), <i>in vitro</i>.<h4>Methods</h4>Platelets (10×10⁷ platelets/mL) from Holstein dairy cows were incubated for 0.5 h or lysed to obtain the PCM and platelet lysate (Lysate), respectively. PMNs were stimulated with PCM for 3h. While BEECs were exposed to PCM or Lysate for 24 h. Real-time PCR was performed to detect the expression of targeted genes (cytokines), including <i>TNFA</i>, <i>IL1B</i> and <i>PGES1</i>. Lipoxin A4 (LXA4; anti-inflammatory mediator) and PGE2 concentrations in the supernatants of PMNs cultured with PCM were measured via ELISA. Cell proliferation in BEECs was assessed using the Cell Counting Kit-8 (CCK-8). Additionally, uterine explants were prepared and processed for immunofluorescence to determine the expression of the LXA4 receptor.<h4>Results</h4>In PMNs, platelet secretions downregulated the mRNA expression of pro-inflammatory cytokines (<i>TNFA</i> and <i>IL1B</i>) and increased LXA4 production. In both PMNs and BEECs, platelet secretions upregulated <i>PGES1</i> expression and PGE2 production. In BEECs, platelet secretions and Lysate upregulated <i>TGFB1.</i> While Lysate suppressed <i>IL1B</i> mRNA expression. Further, platelet secretions showed an anti-proliferative effect in BEECs and increased the LXA4 receptor protein expression in the endometrial epithelia.<h4>Conclusions</h4>Our findings reveal for the first time that platelet secretions directly act on PMNs and BEECs <i>in vitro</i>, thereby assisting the uterine immune network to shift anti-inflammatory environment toward pregnancy. The present study can explain, in part, the successful applications of platelet derivatives in reproductive medicine.