It is widely accepted that the innate immune system is essential as a dynamic factor that could be both protective and detrimental to the maternal-fetal stasis (3
). Although its role is poorly understood, the relative presence of the TLR system in the placental microenvironment further adds to this complexity. Growing evidence clearly suggests that TLR-mediated activation through endogenous signals may indeed provide pregnancy compatible responses (12
). On the other hand, it is not yet fully understood how the TLR machinery will respond to external signals with origins from intrauterine infections and inflammation, particularly when coupled with intrinsic deficiencies in molecules that are critical to normal pregnancy outcome. Two such scenarios are the bacterial and viral DNA breakdown products represented by unmethylated CpG ODN motifs and double-stranded RNA.
In this study, we have examined the gestational age-dependent consequences of CpG ODN administration in pregnant wild type and IL-10−/− mice. We observed that when injected i.p. on gd6 or gd14, CpG ODN caused rapid fetal resorption or preterm stillbirth in IL-10−/− mice, respectively (). In contrast, a similar low dose or a 10 fold higher CpG ODN dose failed to exert any adverse pregnancy effects in WT mice. CpG ODN-mediated consequences in IL-10−/− mice were TLR-9 specific as an antagonistic CpG ODN ligand rescued pregnancy in response to pathogenic CpG ODN (). These observations point to a protective role of IL-10 at the maternal-fetal interface when challenged with bacterial or viral pathogens that may give rise to CpG-like breakdown products.
Several key features were noted in pregnant IL-10−/− mice when challenged with the pathogenic CpG ODN motif. First, the induction of fetal resorption or preterm birth was rapid and severe (). In comparison, WT mice only experienced teratogenic effects at a very high dose (~400 µg/dam) in a portion of the litter at term. Second, the functional presence of uterine CD11b+/F4/80+ macrophages and CD11b+/GR1+ neutrophils (), not CD11c+ dendritic cells or NK1.1+/CD3− NK cells, was significantly increased. This influx was critically associated with a drastic increase in serum and the local presence of mKC (). Uterine macrophages, not GR1+ neutrophils, exhibited significantly high production of TNF-α (). Importantly, both cell types uncharacteristically migrated to the placental zone (). Finally, depletion of F4/80+ cells () or in vivo neutralization of TNF-α () in CpG-treated IL-10−/− mice blunted fetal resorption and preterm birth and rescued pregnancy to term ().
It is important to mention that depletion of GR1+
cells did not lead to rescue of pregnancy nor did it reduce the presence of TNF-α -producing uterine cells (). Thus, when encountered in an abortion- or preterm birth-prone environment such as IL-10 deficiency, TLR-9-triggered cytotoxic activation of macrophages and TNF-α production has severe pathologic effects on pregnancy. Neutrophils may influence pregnancy outcome mainly through production of reactive oxygen species (). These results are important in light of the numerous reports on the association of bacterial and viral infections with early and late pregnancy loss in humans (1
). In connection with these data, recent cohort studies have demonstrated that women with abnormally low IL-10 production are more susceptible to systemic and intrauterine infections, leading to increased incidence of pregnancy loss (49
TLR-9, localized in intracellular compartments, is expressed in both innate and adaptive immune cells and has been shown to be present in uterine immune cells (11
). Curiously, human placental explants did not show any response to CpG compared to agonists for other TLRs (12
), suggesting that in humans the maternal immune cells are the major target for CpG-mediated effects. It is possible that TLR-9 remains localized to the endoplasmic reticulum of the trophoblast in a non-functional form, whereas it becomes activated by localizing to and undergoing cleavage in endolysosomes in uterine macrophages and neutrophils (53
). TLR-9 signaling has been shown to be essential for recruitment and activation of NK and dendritic cells in many settings of parasitic and bacterial infections (55
mice have been shown to lack cytotoxic activation of these cells following infection with Leishmania
). Since we did not observe cytotoxic activation of uNK cells, it is possible that these cells behave differently in response to pathogenic TLR-9 agonists than circulating NK cells.
Our findings of high levels of mKC are critical for implications toward understanding the balance between maternal and fetal health. IL-8 has been shown to be imperative for proper trophoblast cell invasion in humans (6
). On the other hand, dysregulated production of this chemokine in response to a TLR-9-mediated inflammatory insult could prove to be highly detrimental by way of the robust recruitment of cytotoxic macrophages and neutrophils.
We have shown an increased incidence of two cellular populations in response to CpG-mediated pathogens in IL-10−/−
mice, namely macrophages and neutrophils ( and ). While the response of these cells to this TLR-9 ligand is widely expected (57
), this is the first time it has been documented in the context of pregnancy and the uterine microenvironment. We have elucidated novel mechanisms involving these two cell types as first responders to CpG-mediated inflammatory insult, and we have parsed apart their individual roles in this system. Importantly, we have linked the role of macrophages with the production of TNF-α in response to CpG challenge during pregnancy (). Based on our recent findings with other TLR ligands (9
, unpublished observations), we propose that TNF-α is a common final product in response to a spectrum of TLR agonists that induce adverse pregnancy outcomes in response to systemic administration. As shown in and , neutralization of TNF-α resulted in rescue of pregnancy to term suggesting that renewed focus should be assigned to the cellular producers of this cytotoxin in response to a particular TLR that is activated. An understanding of the cellular diversity of TNF-α production at the maternal-fetal interface may be essential in providing proper therapeutic interventions in the context of pregnancy complications.