Evaluation of Sars-CoV-2 entry receptors in COVID-19 positive and negative placentae: correlation between expression and infectivity

Problem: The risk of pre-term delivery, pre-eclampsia (PE), cesarean section, severe neonatal/perinatal morbidity and mortality was significantly increased in COVID-19 pregnant women (2). The infection of host cells by SARS-CoV-2 requires the presence of two receptors: the transmembrane receptor Angiotensin-Converting Enzyme 2 (ACE2) and the Type II Transmembrane Serine Protease (TMPRSS2) (3,4). Moreover, SARS-CoV-2 may bind to a third alternative receptor, the cluster of differentiation 147 (CD147)/EMMPRIN (5). There is therefore a lack of information on how maternal infection with SARS-CoV-2 impacts the expression of ACE2 and TMPRSS2 in the placenta, at both transcript and protein level. Here, we investigated term placentae collected before the SARS-CoV-2 pandemic and COVID-19+ placentae, analyzing the expression of the three main COVID-19 receptors, namely ACE2, TMPRSS2 and CD147. Method of Study: We performed RT-qPCR, Western blot and cytofluorimetric analysis on placental tissues and on isolated placental cells, both in resting conditions and after stimulation with Spike protein. Finally, we evaluated the SARS-CoV-2 capability to infect placental cells in vitro. We observed very low expression of the canonical SARS-CoV-2 entry receptors on syncytio- and cytotrophoblast cells, as well as on human umbilical vein endothelial cells (HUVECs), but the infection by the virus resulted into an up-regulation of their expression. In order to try to understand the mechanisms underlying the resistance to Sars-CoV-2 infection of placental cells, we performed transfection tests with the Spike protein to see the formation of cellular syncytia. The simultaneous presence of Spike and functional receptors (ACE2, TMPRSS2) on the cell surface, in fact, leads to the activation of fusogenic mechanisms, which manifest with the formation of syncytia. Results: Our results lead us to affirm that in healthy placentas there are elevated levels of CD147 expression and variable levels of ACE2, while TMPRSS2 is not present, identifying placenta as a non-preferential tissue for Sars-CoV-2 infection in normal conditions. The COVID-19 syndrome and the presence of circulating virus could determine an up-regulation of TMPRSS2 facilitating the virus to enter the syncytiotrophoblast and cytototrophoblast cells. Conclusions: We have demonstrated that endothelial cells are not permissive for virus entry, but the systemic effect linked to endothelitiis and therefore to the preeclamptic-like syndrome, could be due to the induced direct activation of the Spike protein (with its shedding and release) on placental cells, in terms of increased expression of proinflammatory cytokines, increased vascular permeability and induced syncytiotrophoblasts apoptosis. Finally, because the transfection of placental cells with Spike protein and the simultaneous overexpression of ACE2 did not lead to cell fusion, an intrinsic mechanism of cell fusion resistance expressed by placental cells, is proposed.