It is well known that in the placenta, IEs adhere and activate the ST through CSA interaction [12, 15]. In this study an in vitro placental sequestration model was established using non-syncytialized BeWo trophoblasts cellular line, in order to investigate its response to the parasite binding and to TNF. The findings suggest that BeWo cells responded to CSA-IEs and TNF, promoting a pro-inflammatory environment in the placenta. This suggestion based in the increased mICAM-1 expression and secretion of immune factors, such as IL-8, IL-6 and sICAM-1.
It is important to highlight that the BeWo cell line supports the adherence of IEs via CSA, and it is a useful model for studying parasite-host interactions in placental malaria [10–13]. In agreement with previous reports, it was found that BeWo cells express a receptor profile similar to primary placental ST, characterized by the constitutive expression of surface CSA and ICAM-1, and the absence of CD36 on the cellular surface [11–13]. It is known that P. falciparum adheres to ST via CSA, therefore, strains were subjected to CSA panning to make parasites adherent to BeWo cells. After panning the selected parasites adhered to CSA confirming the previous findings showing that parasites with different genetic repertoires can be selected in vitro to adhere to CSA . Differences in parasite adherence to BeWo cells were found among the four CSA-strains with the highest adherence observed in FCB1csa strain. The different adhesive profiles observed between strains with different geographical origin and genetic repertoire could be explained by PfEMP-1 molecular and structural characteristics, codified by highly variable genes or by specific VAR2CSA expression levels on the IEs surface.
In the adherence model BeWo cells response to CSA-IEs and TNF stimuli, both implicated in placental malaria pathogenesis . TNF or CSA-IEs of P. falciparum increased surface expression of ICAM-1 and appear be related to the adherence ability of IEs because the more adherent lines (FCB1csa and 3D7csa) had a significant effect. Moreover, it was found that the combination of stimuli (resembling the physiological scenario of placental malaria) causes a greater increase in this adhesion molecule levels. ICAM-1 receptor recognizes its ligand (LFA-1 and MAC-1) within immune system cells and promotes their recruitment to inflammation sites . The increase in this receptor’s expression on the ST’s surface is a characteristic of placental inflammatory disorders and promotes immune cells adhesion to ST, augmenting local immune responses.
In vitro studies with primary ST cultures suggest that cytokines, such as TNF and/or IFNγ stimulate monocyte-ST adherence through an ICAM-1 increase [18–20], this interaction is important from a pathogenic point of view, since monocytes recruitment can result in ST cell apoptosis and disruption of this barrier in presence of TNF . It has been shown that infectious agents associated to placental inflammatory conditions regulate ICAM-1 in the ST, trophoblasts HIV-1 or cytomegalovirus infection, such as the contact of ST with Toxoplasma gondii-infected mononuclear cells, leads to an over-expression of ICAM-1 and promotes an increase of monocytes and lymphocytes adhesion in the ST [20–22]. Supporting these observations, the immunohistochemical analysis of placentas with placentitis by T. gondii and Trypanosoma cruzi shows an ICAM-1 over-expression in the ST associated to leukocyte infiltrations and trophoblastic barrier loss . Finally, ICAM-1 hyper-expression has been reported on P. falciparum-infected placentas .
It was observed that upon contact with IEs and haemozoin, the ST releases different cytokines and chemokines [14–16]. Ex vivo models support the role of foetal cells in the inflammatory response to malaria infection [4, 8]. Release of IL-8, IL-6, sICAM-1, MIP-1α, MCP-1 and TNF from BeWo cells under TNF and IEs-CSA stimulation was investigated and an increase of IL-8, IL-6 and sICAM-1 concentration was found. Less adherent strains (FCR3csa and FCB2csa) induced lower release levels of these cytokines, particularly in IL-6 and sICAM-1, which may suggest that the more adherence of IEs, the more inflammatory reaction. Luchhi et al. reported that following IEs interaction, ST increases the mRNA expression of IL-8, the activation of Jun N-terminal kinase 1 (JNK-1) and the secretion of MIF and MIP-1α . Haemozoin stimulated ST cells release molecules, such as IL-8, MIP1a, and MIP1b and sICAM-1 . Reports have been shown that ex vivo explants of foetal tissue from malaria-positive placentas secreted significantly amounts of IL-6  and IFN-γ  compared to uninfected placentas and its levels has been associated with low birth weight . These findings suggest an inflammatory reaction in the trophoblastic cells, which promotes maternal leukocytes recruitment in the placenta’s surface and then augments inflammatory molecules production.
Plasmodium falciparum placental infection is characterized by mononuclear infiltrates in the IVS and local production of inflammatory cytokines and chemokines produced by maternal and foetal cells which increase the arrival and accumulation of mononuclear cells [8, 9, 32]. TNF increased levels and accumulation of monocytes in the IVS have been associated to low birth weight [3, 5, 6, 32]. Additionally, it has been reported that malarial placentas express higher IL-8 levels, which are associated with placenta leukocytes density and intra-uterine growth delay . Similarly, Fievet et al. reported increased IL-6 production in cultures of infected placental villi .
An increase in soluble form of ICAM-1 receptor (sICAM-1) was found in supernatant cultures of BeWo cells in response to IEs and TNF. These results suggest that the BeWo cells activation by inflammatory (TNF) or IEs adherent to CSAs results in a mICAM-1 over-expression and in its release from cellular surface to its soluble form or production. It is hypothesized that the ICAM-1 release as a protection mechanism in order to regulate the local inflammatory response [28, 33], through to reduce mICAM-1 levels and as consequence the leukocytes adherence, to compete with the membrane-anchored form by the LFA-1 and Mac-1 ligands expressed in leukocytes, which prevents the adherence of these cells to the cellular surface  and to contribute to the production of inflammatory molecules [28, 35].
Harwell et al. found high blood levels of sICAM-1 in P. falciparum-infected placentas compared to non-infected placentas’ levels. They also observed a positive association between newborns’ weight to multigravidae women with placental malaria, suggesting that during the infection sICAM-1 released from the placental cells membrane may have an immunoregulatory purpose, in order to reduce load of immune cells in the placenta .
Co-culture of late trophozoites and schizonts with non-syncytialized BeWo incubated for up 16–18 hours may have some drawbacks. During parasite maturation schizonts ruptures and release haemozoin, glycosylphosphatidylinositol (GPI) and other parasite-derived products, therefore, it is possible the unspecific stimulation in the BeWo cell model. To determine specific parasite-BeWo activation, short incubation times and unselected-CSA parasites may shed light on parasite-trophoblast interaction/activation. Removing surface adhesive proteins from IEs surface by trypsinization or blocking adherence with soluble CSA should help to establish a relationship among cytoadherence and cell activation. Non-syncytialized BeWo cells showed cell response and adhesion patterns, however as BeWo cells were not syncytialized then it should be considered for further adherence and cytokine expression experiments.