Intrauterine infections are a well-established cause of preterm labor and have been linked to other pregnancy complications, such as preeclampsia and intrauterine growth restriction (1
). However, the mechanisms involved are still largely undefined. A key observation in a number of abnormal pregnancies is that during early pregnancy, trophoblast cell apoptosis is significantly elevated (11
). The findings of this current study suggest that an intra-uterine Gram-positive bacterial infection may cause the excessive trophoblast cell apoptosis observed in some abnormal pregnancies and that TLRs may provide the mechanism of pathogenesis. Specifically, our results demonstrate that Gram-positive bacterial PDG causes trophoblast apoptosis in vivo. Our in vitro studies implicate TLR1 and TLR2 in providing the direct mechanism of PDG-induced apoptosis which can be regulated by TLR6.
A number of animal models have been used to test the effects of infection on pregnancy. However, the majority of these studies have focused on Gram-negative bacteria or bacterial components (52
). Two studies in mice have evaluated the effects of Gram-positive bacterial components on pregnancy outcome. Kajikawa et al. (41
), found that i.p. administration of LTA on embryonic day 15 resulted in preterm delivery and trophoblast apoptosis. This is consistent with our previous in vitro observations that LTA can induce first trimester trophoblast apoptosis (40
). In another study, i.p. administration of a high dose of PDG on embryonic day 14.5 was also found to trigger preterm delivery (42
). However, contrary to another stimuli, such as bacterial LPS, which induces preterm delivery in association with elevated cytokine production and changes in intrauterine immune cell activation and distribution (44
), the effect of PDG was not associated with changes in cytokine production (42
). We have also previously reported that PDG induces apoptosis in human first trimester trophoblast cells (40
), suggesting that preterm labor observed in these animals may be mediated by PDG-induced trophoblast cell death. Since a high PDG dose of 1 mg per mouse given on day 14.5 of gestation (embryonic day 14.5) triggers rapid prematurity (42
) and pregnancy complications are thought to be established early in pregnancy (1
), a lower PDG concentration was selected and administered to mice early in gestation, so that prematurity would not be induced and the mechanisms involved could be assessed. Indeed, delivery of 25 μ
g of PDG on embryonic day 6.5 did not cause preterm delivery nor fetal resorption. In addition, there was no evidence of changes in uterine NK cell and myeloid cell levels or uterine NK cell cytotoxicity, as seen in other models (44
). Instead, delivery of this low dose of PDG resulted in excessive trophoblast apoptosis, evidenced by M-30 and active caspase 3 immunostaining, as well as elevated placental caspase 3, caspase 8, and caspase 9 activities. Furthermore, high levels of apoptosis within the fetal membranes were observed. This latter observation may explain why in vivo, PDG causes preterm delivery rather than other pregnancy complications, and in humans this may manifest as preterm labor associated with membrane rupture (55
). As mentioned above, unlike the LPS-induced models of preterm labor (44
), delivery of PDG failed to induce an inflammatory response at the maternal-fetal interface. There were no detectable differences in placental cytokine and chemokine levels, nor was there any significant decidual immune cell infiltrate. These findings suggest that placental apoptosis may provide the primary mechanism by which preterm labor is triggered in vivo by Gram-positive bacterial PDG.
Having established that PDG was indeed triggering trophoblast apoptosis in vivo, the next objective of this study was to determine the cellular mechanisms involved. As mentioned before, we have previously reported that first trimester trophoblast cells, which express TLR2 and TLR1, but lack TLR6, undergo apoptosis following exposure to the TLR2 ligands PDG and LTA (40
). In contrast, the TLR4 ligand LPS does not induce trophoblast apoptosis, but instead triggers an inflammatory response (40
), indicating that this proapoptotic effect is specific for TLR2. Unlike other TLRs, TLR2 can cooperate with either TLR1 or TLR6 (35
). This heterodimerization has been shown to allow TLR2 to discriminate between ligands, particularly with respect to lipoproteins and lipopeptides. For example, TLR2/TLR1 dimers can recognize tri-acylated lipopeptides, while TLR2/TLR6 dimers recognize diacylated lipopeptides (36
), and differences in the extracellular domains of TLR1 and TLR6 are responsible for this ligand specificity (63
). However, in some cases, usage of TLR1 and TLR6 appear to be more relevant to function rather than ligand specificity. TLR2 activation by S. aureus
or its components is enhanced in the presence of TLR6 and inhibited by TLR1 (35
). Our results indicate that the type of TLR expressed by the trophoblast may determine the type of response generated. Thus, the proapoptotic effect appears to depend on the presence of TLR2 and TLR1, since inhibiting the signaling potential of either receptor decreases PDG-induced apoptosis. These results correlate with our previous findings where we demonstrated that PDG triggers first trimester trophoblast apoptosis through activation of the caspase pathway in a Fas-associated death domain protein-dependent manner (40
). Similar results have been shown in other cell types, where bacterial lipoproteins are able to trigger TLR2-mediated apoptosis through the recruitment of Fas-associated death domain protein by MyD88 (22
). Furthermore, this induction of apoptosis is independent of the NF-κ
B pathway, since we did not observe either NF-κ
B activity or cytokine production. In a previous study, we evaluated the cytokine/chemokine content of the trophoblast (cell lysates) 12 h after treatment with a low PDG dose and found an increase in the intracellular levels of IL-8 and IL-6. For this we analyzed the trophoblast cell lysates after treatment with a low PDG dose (10 μ
g/ml) for 12 h (40
). However, when we looked for changes in IL-8 and IL-6 at later times, we observed a different response. In the current study, we evaluated the cytokine content and secretion by the trophoblast following a 24- and 48-h treatment at low, moderate, and high PDG concentrations. Contrary to what we observed at 12 h, at these later time points, we saw both IL-8 and IL-6 content (lysates) and secretion (supernatants) inhibited, which correlates closely with the 24- to 48-h induction of caspase activation. We could hypothesize that the early increase in IL-8 and IL-6 content may represent an additional effort of the cell to prevent apoptosis, which at later time points becomes overridden by the proapoptotic event.
Having established the role of TLR1 and TLR2 in PDG-induced trophoblast apoptosis, we next questioned what effect the presence of TLR6 would have on the trophoblast response to PDG by introducing functional TLR6 into wild-type trophoblast cells. In the presence of TLR6, PDG-induced trophoblast cell death was inhibited. Furthermore, the presence of TLR6 was able to reverse the effect of PDG in terms of NF-κ
B activity and cytokine production. These data indicate that in response to PDG, TLR2/TLR1 dimers mediate trophoblast apoptosis, while TLR2/TLR6 dimers promote trophoblast survival, but instead triggers cytokine/chemokine production. In support of this, Nakao et al. (60
), have demonstrated that PDG-induced cytokine production in monocytes is generated via TLR2 and TLR6, but not through TLR1 (60
). TLR6 may have a greater affinity for PDG and, therefore, may be able to compete with TLR1 for ligand binding. Alternatively, the survival signals triggered by PDG through TLR2/TLR6 may outweigh the pro-apoptotic signals generate by TLR2/TLR1 (66
Although the first trimester trophoblast cell lines used in these studies lack TLR6, term placental tissue has been shown to express TLR6 mRNA, albeit at low levels (67
). However, the cellular localization of TLR6 is currently unclear, as is the expression levels and distribution earlier in gestation. Nonetheless, studies have shown that TLR6 expression in myeloid cells can be up-regulated by IL-6 and LPS and down-regulated following exposure to whole Gram-negative bacteria (67
). Thus, the microenvironment at the implantation site my have a powerful influence on the expression of TLRs (68
). In the case of TLR6, its expression by the trophoblast may determine whether there is an active immune response to the bacterial infection or placental cell death triggered by the same microbe. Furthermore, in the presence of TLR6, a change in the placenta’s response to PDG from apoptosis to NF-κ
B activity and inflammation may have significant physiologic consequences. Instead of cell death-triggered prematurity, the inflammatory response may act to facilitate resolution of the infection and protection of the pregnancy. However, if too strong, the TLR2/TLR6-mediated inflammation may itself trigger an adverse pregnancy outcome.
In summary, we have demonstrated, using an animal model, that PDG induces trophoblast apoptosis, suggesting that elevated placental cell death may provide the mechanism underlying PDG-induced prematurity. Using a human in vitro model, we have shown that PDG-induced trophoblast apoptosis is mediated by TLR2 and TLR1 and can be reversed by the presence of TLR6. Furthermore, TLR2/TLR6 responses to PDG results in trophoblast NF-κB activation and cytokine/chemokine production. The findings of this study suggest that a Gram-positive bacterial infection, through TLR2 and TLR1, may directly promote the elevated trophoblast cell death observed in a number of pregnancy complications. Together, these findings suggest that in the trophoblast the expression of TLR6 is a key factor determining whether the response to PDG would be apoptosis or inflammation.