Congenital Chagas disease, caused by Trypanosoma cruzi (T. cruzi), is partially responsible for the increasing globalization of Chagas disease despite its low transmission. During congenital transmission, the parasite reaches the fetus by crossing the placental barrier. However, the success or impairment of congenital transmission of the parasite is the product of a complex interaction between the parasite, the maternal and fetus/newborn immune responses and placental factors. There is other evidence apart from the low congenital transmission rates, which suggests the presence of defense mechanisms against T. cruzi. Thus, the typical amastigote nests (intracellular parasites) cannot be observed in placentas from mothers with chronic Chagas disease nor in human placental chorionic villi explants infected in vitro with the parasite. In the latter, only a few parasite antigens and DNA are identified. Accordingly, other infections of the placenta are not commonly observed. All these evidences suggest that the placenta can mount defense mechanisms against T. cruzi.
Immune response during pregnancy
Mammalian pregnancy poses a unique challenge to the maternal immune system; tolerance needs to be maintained in the semi-allogeneic fetus while effectively retaining immune reactivity to protect the mother and fetus from the deleterious effects of infections [1,2]. During pregnancy, the maternal immune system is characterized by a reinforced network of cellular and molecular recognition, communication, trafficking, and repair; it raises the alarm to maintain the health of the mother and the fetus. On the other hand, the fetus provides a developing active immune system that modifies the way the mother responds to the environment, creating a unique pattern of immune system responses . The capacity of the mother and fetus/newborn to mount innate and specific immune response(s) against pathogens determines whether a fetal/neonatal infection might be prevented, limited, or permitted [4,5]. Activation and maturity of the maternal/fetal immune system are fundamental to prevent Trypanosoma cruzi vertical transmission. It has been shown that in uninfected babies, born to infected mothers, the production of pro-inflammatory cytokines increases . Contrarily, the levels of inflammation markers and activation of NK cells are rather low in congenitally infected newborns . On the other hand, maternal T. cruzi-specific IgG antibodies play protective roles in mothers and fetuses when antibodies are transferred through the placenta and also may contribute to a reduction in parasitemia .
Importantly, the placenta, as an immune regulatory organ, acts as a modulator of fetal and maternal immune responses . Therefore, the current concepts of the immune response during pregnancy are changing. The theory, which was first proposed by Sir Peter Medawar  that the implanting embryo is a semi-allograft and is capable of triggering a maternal immune response that could mediate its rejection, is now outdated .
The placenta and its role in the defense against T. cruzi
The human placenta is the primary barrier between the maternal and fetal compartments throughout pregnancy. The exact defense mechanisms of the placenta to limit pathogen access to the fetus are not completely understood. However, the trophoblast plays a major role in the antiparasite defenses, forming part of the innate immune response. Three types of defense mechanisms in innate immunity have been described: (i) anatomical barriers, such as the placental barrier (Figure 1); (ii) cellular innate immune responses; and (iii) humoral innate immune responses. During tissue invasion, pathogen breaks the anatomical barriers, and innate immune cells are activated and secrete cytokines and chemokines to control pathogen replication [9,10]. The placental barrier is part of these innate immune defenses.
The maintenance of the trophoblast at the placental anatomical barrier.
Trophoblast epithelial turnover as a mechanism against T. cruzi infection
The epithelial turnover is considered to be a protective mechanism against pathogens in different organs, such as the skin and urogenital tracts [11,12]. There is a basal level of epithelial renewal, which can be accelerated or decreased in response to various stimuli. Infection usually stimulates epithelial turnover . The normal epithelial turnover is one mechanism that assures the integrity of the anatomical barriers. Moreover, the epithelial turnover has been considered part of the innate immune system, because pathogens, before cell invasion, must attach to the superficial cells. As these cells are continuously eliminated, the attached pathogens are removed with them .
The trophoblast is a lining epithelium that is in contact with the maternal blood and therefore the first fetal tissue in contact with circulating infective trypomastigotes. The trophoblast is composed of two cellular layers: the syncytiotrophoblast (ST) and the cytotrophoblast (CT). The CT displays high proliferative properties, whereas the differentiated ST loses its generative capacity and is no longer able to proliferate. The ST is a multinucleated layer that forms the outer surface of placental villi which is generated and maintained through syncytial fusion by the incorporation of CT cells . The continuous incorporation of CT cells into the ST is counterbalanced by the formation of apoptotic ST knots, which are released into the maternal blood. We propose that the trophoblast turnover should be considered as a defense mechanism against pathogens since we have previously shown that T. cruzi: (i) increases proliferation markers such as PCNA and Ki-67 and enhances DNA synthesis in the BeWo trophoblastic cell line ; (ii) induces trophoblast differentiation markers β-hCG and syncytin and cell fusion in human placental chorionic villi explants (HPCVE) and BeWo cells ; (iii) induces apoptotic cell death in HPCVE and BeWo cells, verified by DNA fragmentation and by caspase-3 and caspase-8 activations [15,16].
Placental innate immune cellular and humoral responses against T. cruzi
The innate immune response against pathogens is initiated by pathogen pattern recognition receptors, which include Toll-like receptors (TLRs) that recognize and bind highly conserved sequences known as pathogen-associated molecular patterns. The human trophoblast expresses all 10 of the known functional TLRs , and T. cruzi is recognized by TLR-2, TLR-4, TLR-7, and TLR-9. Surface TLRs (TLR-2 and TLR-4) recognize glycosylphosphatidylinositol-anchored mucin-like glycoproteins from T. cruzi surface [18–20]. We have shown that T. cruzi infection is related to TLR-2, but not to TLR-4 and TLR-9, expression and activation . The binding of TLR-2 to its ligands leads to activation of signaling pathways and up-regulation of genes involved in the innate immune response including cytokines and chemokines [17,19]. T. cruzi induces the secretion of IL-1β, IL-6, IL-8, IL-10, and TNF-α in HPCVE . Interestingly, IL-1β, IL-6, and TNF-α secretion is also associated with cellular proliferation and differentiation in the trophoblast [21,22] and inhibition of TLR-2 impairs trophoblast turnover (manuscript under review in ‘Placenta’). However, until now, we do not know whether the activation of TLRs occurs mainly in the trophoblast or if other placental cells are also involved, this matter should be addressed in the future. Importantly, as a consequence of our results, the TLR-2-initiated cytokine profile should also be considered as a local placental defense mechanism.
Other possible placental defense mechanisms
The placenta, and particularly the trophoblast, expresses many non-coding RNAs including microRNAs (miRNAs) that regulate placental development function. Moreover, different miRNAs exhibit specialized functions during normal and pathological pregnancies. Placental miRNAs, packaged within exosomes and other vesicles or bound in protein complexes, are capable of communicating distinctive signals to maternal and fetal tissues . Placenta-specific and trophoblast-derived miRNAs, encoded in the chromosome 19 miRNA cluster (C19MC) and released within exosomes, resist viral infection in other mammalian cells . Preliminary results from our laboratory have shown that T. cruzi induces in HPCVE a specific C19MC-encoded miRNAs profile. Some of those miRNAs are involved in the regulation of immune functions, particularly those of TLR-mediated pathways . Studies on T. cruzi-induced miRNAs and exosomes are currently ongoing and are of particular interest since miRNA pathways are potential diagnostic tools and targets for therapeutic control of parasitic diseases  and other pathologies, including placenta-derived ones . Targeting miRNAs constitute a promising possibility for the treatment of different diseases due to the following reasons: (i) miRNAs are regulators of gene expression, (ii) are relatively easy to manipulate, (iii) can be administrated in vivo, and (iv) present an apparent lack of adverse effects when administered intravenously. Moreover, miRNAs are detectable in biological fluids, thus offering real potential as non-invasive biomarkers, providing new diagnostic and therapeutic options during pregnancy and for several diseases as well [27,28].
In summary, the studies about the placental defense mechanism that determines the probability of infection, together with parasite, maternal, and fetal/newborn factors, are of outstanding interest since they are potential diagnostic, prognostic, and therapeutic tools.
During congenital transmission, Trypanosoma cruzi reaches the fetus by crossing the placental barrier.
Evidence suggests that the placenta can mount defense mechanisms against T. cruzi.
Epithelial turnover of the trophoblast, parasite-induced specific cytokine profile and miRNA profile should be considered as a local antiparasitic immune response.
The Authors declare that there are no competing interests associated with the manuscript.