Epidemiologic studies have reported an association between viral infection either in the genital tract or in the non-genital tract, and preterm labor.28–32
However, the potential mechanism by which viral infection might induce preterm labor has not been elucidated. In the present study, we demonstrated that intraperitoneal injection of Poly[I:C], a synthetic viral dsRNA, induced preterm birth in mice. Furthermore, we demonstrated that the placenta, and more specifically the trophoblast, plays an active role in the response to Poly[I:C] through the production of cytokines and chemokines, and this response is mediated through TLR3 expression and function.
Bacterial and viral infections pose a significant threat to pregnancy and well-being of the fetus by gaining access to the placenta through one of three major routes: by way of the maternal circulation; by ascending into the uterus from the lower reproductive tract or by descending into the uterus from the peritoneal cavity.33
Clinical studies have established a strong association between pregnancy complications and intrauterine infections.33–35
Indeed, infections have been reported as responsible for up to 40% of preterm labor cases. Furthermore, 80% of preterm deliveries occurring at less than 30 weeks of gestation have evidence of infection.34–36
In addition, other pregnancy complications, such as pre-eclampsia, may have an underlying infectious trigger.37–39
One of the main questions of this study was how a microorganism, in this case a virus, might initiate a response that would induce preterm labor or abortion, or even pre-eclampsia.
dsRNA is a universal viral PAMP produced by most viruses at some point during their replication40
and it is recognized by intracellular TLR3 expression. The response following binding of TLR3 is characterized by cytokines and chemokines that will mount active immune responses. We found that Poly[I:C] has a striking effect on the survival of pregnancy by inducing preterm labor in less than 24 hr following treatment. These results confirmed previous studies, which demonstrated that intrauterine injection of Poly[I:C] induced preterm birth.41
However, the doses of Poly[I:C] used in those studies were very high which could affect the well-being of the mother and therefore affect the fetus. In this study, we used a dose that had no effect on the maternal well-being; therefore, the outcome of the pregnancy would be the result of changes at the maternal-fetal interface.
Interestingly, the dose used in this study for Poly[I:C], which was able to induce preterm birth without inducing any maternal systemic adverse, is exceptionally lower (4.5 mg/kg) compared with 30 mg/kg previously reported for dsRNA induced disease in mice.42
To characterize the mechanism by which Poly[I:C] induced preterm delivery, we evaluated histologic changes at the feto-maternal interface in our mouse model. We observed a massive inflammatory process characterized by infiltration of immune cells (polymorphonuclear cells and NK cells), necrosis, hemorrhage and edema, mainly localized in the placenta, the amniotic membrane, and the umbilical cord. Some of these morphologic characteristics are observed in cases of human chorioamnionitis and funisitis.
At the normal murine feto-maternal interface, immune cells such as neutrophils, macrophages and NK cells are assumed to be excluded from the placenta and localized only in the decidua.12
Treatment with Poly[I:C] disrupt this normal distribution and induce a massive migration of immune cells, primarily NK cells from the decidua towards the placenta, invading the spongiotrophoblast and then the labyrinth. This type of migration is consistent with an immune response, which would exert adverse effects on pregnancy such as fetal demise or abortion.43
Interestingly, macrophages followed a different migration pattern; we did not observe any change in macrophage distribution at the decidua-placenta interface; however, we found a significant number of macrophages at the fetal side of the placenta. These results suggest that the fetal side could actively contribute to mounting an immune response that could endanger the fetus. Indeed, a recent study in humans described that in cases of villitis of unknown etiology (VUE), placentas from male neonates showed CISH+ signals from Y chromosomes in a majority of macrophages, but not in lymphocytes, indicating that the macrophages were of fetal origin.44
Therefore, we could hypothesize that a viral infection of the placenta could be the trigger for the migration of fetal macrophages.
The pattern of cytokines and chemokines observed in the placental tissue following Poly[I:C] treatment suggests that the placenta plays an active role in the response to viral infection. Indeed, the pattern correlates with the histologic findings. Chemokines, such as KC and G-CSF, are known to attract neutrophils; IL-12p40 traffics macrophages, MCP-1, MIP-1α and MIP-1β attract macrophages and NK cells. All these cytokines were significantly increased in their expression levels within the placenta upon Poly[I:C] administration. These chemokines may be responsible for attracting immune cells from either the maternal side or the fetal side towards the placenta.
We also found a significant increase in the levels of pro-inflammatory cytokines in the placenta following Poly[I:C] treatment. Pro-inflammatory cytokines such as IL-6, TNF-α
are known to be elevated in amniotic fluid or amnion/choriodecidua membrane from women with preterm labor.45–48
Pro-inflammatory cytokines are believed to regulate intrauterine prostaglandin production, which is an important aspect of the onset of labor in terms of inducing uterine contraction and cervical ripening.49
They also increase the levels of matrix metalloproteinases (MMPs) expression and activity in the membranes50
and may induce apoptosis of trophoblast cells.51
Therefore, it is plausible to conclude that the necrotic areas and damage observed in the placenta and membranes of mice treated with Poly[I:C] may be the result of the high levels of these pro-inflammatory cytokines.
Our next question was whether the trophoblast could be the origin of the signals controlling the migration of these immune cells. Recently, we proposed that the trophoblast functions as an immune regulator and has the capacity to influence, based on the type of signals, the differentiation and migration of immune cells.13
Indeed, we have shown that trophoblast cells can induce the migration of macrophages and NK cells14
and modulate macrophage response to bacterial products such as LPS.24
Our in vitro
study demonstrated that upon Poly[I:C] stimulation, either mouse primary trophoblast or human trophoblast showed cytokine/chemokine production profiles similar to in vivo
results, suggesting that trophoblasts were primarily responsible for the response to this PAMP, although we cannot exclude the possibility that other cell types present in the placenta may also contribute to this response. This finding supports the notion that trophoblasts play a role in coordinating the maternal innate immune response to infection at the feto-maternal interface12,52
and, especially in this case, in response to viral infection.
In terms of the molecular mechanism of action, we have demonstrated a critical role for TLR3 on Poly[I:C] induced pre-term labor. In contrast to wt mice, TLR3KO mice did not undergo preterm delivery following Poly[I:C] administration, clearly suggesting that TLR3, and the concomitant cytokine response, is required for the in vivo
response to Poly[I:C] and inducing preterm delivery. Cytokine/chemokine production upon Poly[I:C] injection was also impaired in TLR3KO mice, with some exceptions such as IL-6, which showed a slight increase by Poly[I:C] in TLR3KO mice. This is consistent with recent findings that showed the presence of a TLR3 independent, MDA5 dependent recognition of Poly[I:C] for regulating IL-6 production.53
We also confirmed that dsRNA ligated TLR3 and activated the NF-κ
B signaling pathway, inducing cytokine/chemokine production in vivo
and in vitro
using human trophoblast cells.
In summary, we have demonstrated that systemic administration of dsRNA induced preterm delivery through TLR3 activation in a mouse model. We show that in addition to the classical immune response mediated by the maternal immune system, the placenta, and more specifically the trophoblast, is able to recognize, through TLR3, the presence of dsRNA and mount a strong and specific immune response. Furthermore, the trophoblast may not only influence the maternal immune system, but may also induce the migration and differentiation of fetal immune cells. This contribution of the trophoblast in the maternal and fetal innate immune system supports the notion that the trophoblast acts as a component of the innate immune system. Our study raises the possibility of clinical significance of viral infections screening among pregnant or pre-pregnant women to identify a high-risk population for pre-term labor.