Infectious Diseases in Obstetrics and Gynecology 5 Table 1: Routes of pathogenic entry coupled to speciﬁc TLR activations lead to preterm birth via NF-κB-TNF-α -dependent and independent pathways. Route of Entry IU IU Systemic IU Systemic IU Systemic NF-κB − − + TNF-α − − + − ++ − ++ Pregnancy Outcome Preterm Birth11 Preterm Birth11, up Preterm Birth60 Preterm Birthup Preterm Birth13 Preterm Birth/IUFDup Preterm Birth/IUFD15 TLR TLR2 TLR3 TLR3 TLR4 TLR4 TLR9 TLR9 (PAMP) peptidogylcan poly(I:C) poly(I:C) LPS/E. coli LPS/E. coli CpG CpG – up is our unpublished data Recently, it was shown that TLR3 can recognize products of cellular necrosis, a fact of importance for a system such as pregnancy where cellular turnover is abundant [59]. Evidence suggests that TLR3 activation in trophoblasts may be a key mechanism to preterm birth outcome induced by infectious agents [60]. Trophoblasts express the coxsackievirus and adenovirus receptor (CAR). Upon adenoviral infection, trophoblasts underwent apoptosis and this led to recruitment of a decidual immune response [42]. Studies in human and mouse trophoblast cells show that cells were treated with synthetic TLR3 ligand poly(I:C), and the NF-κB pathway was activated leading to downstream cascades of inﬂammatory signals. Studies have utilized poly(I:C) as well to assess the role of preterm birth through TLR3 activation in mouse models. Where poly(I:C) was injected intraperitoneally to mimic a maternal systemic infection, mice gave preterm birth through an NK-κB-dependent mechanism and demon- strated marked serum and placental production of IL- 6, IL-12, and MCP-1 [11, 60]. Our unpublished results demonstrate that preterm birth in WT mice is induced by a TNF-α-mediated axis. In contrast, in a model of intrauterine infusion, INF-β and CCL5 were the major contributors to preterm birth outcomes in response to poly(I:c) and TNF- α production was not signiﬁcant [11]. Taken together, these studies lend evidence to the hypothesis that route of entry of a virus may play a role in the speciﬁcity of the immune response evoked (Table 1). 6.3. TLR4. It is well established that TLR4 recognizes lipopolysacharride motifs found on the majority of gram negative bacteria. Two of the three bacterial strains associated with preterm birth outcomes, mycoplasma hominis and trachtomonas vaginalis, are gram negative and thus have the capacity to signal via TLR4 [46]. Activation of TLR4 at the placental level has been studied in order to assess the possible immune activation that ensues when TLR4 speciﬁc bacterial loads overwhelm the uterine cavity and trigger preterm birth [61]. Our lab has demonstrated that LPS and E. coli cause increased uNK cell cytotoxicity and TNF-α production, ultimately leading to preterm birth. Neutralization of TNF- α or abolition of uNK cells rescued pregnancy to term. In contrast, our unpublished model of E. coli intrauterine infusion demonstrates that preterm birth does occur, but not in a TNF-α-dependent manner ([13], our unpublished data). Similar unpublished data from our lab agree with this data as the NF-κB pathway was not activated in a model of preterm birth mediated by LPS intrauterine infusion (Table 1). Again, these results present strong evidence for the hypothesis that alternative routes of pathogenic entry, for both viral and bacterial pathogens, can cause markedly diﬀerent immune responses, both ultimately leading to preterm birth. 6.4. TLR7/8. TLR7/8 are speciﬁc for single stranded RNA motifs, thus retroviruses are strong candidates to activate these receptors. Though HIV is not yet correlated to a speciﬁc TLR for activation, evidence suggests that TLR7 activation mimics HIV related pathologies [62]. Importantly, strong associations exist between HIV seroprevalance and incidence of preterm birth. A cohort study that followed 600 women, approximately half seropositive for HIV and half seronega- tive, showed that HIV+ women were signiﬁcantly more likely to give preterm birth. Furthermore, the seropositive women who gave preterm birth showed signiﬁcantly diminished levels of CD4+ T cells [63]. Taken together, these data demonstrate that either HIV or the immune-compromised state of mothers may lead to increased risk of preterm birth. Studies in nonpregnant patients aim to activate TLRs in order to discourage HIV activity due to the proinﬂammatory activity of antiretroviral therapy [64]. Taken together, the immunosuppressive state of pregnancy needs to be thor- oughly considered when therapy is employed in pregnant women, but lessons may be learned from how the virus travels and replicates during this state. 6.5. TLR9. TLR9 recognizes unmethylated CpG motifs. Though reports identify this intracellular receptor as speciﬁc for double stranded DNA viruses or fetal DNA, these types of CpG motifs constitute over 80% of bacterial genomes as well. Studies that activate TLR9 with CpG in pregnant mice thus far have demonstrated that a lack of IL- 10 causes high susceptibility to CpG-mediated pathogenic mimics and inﬂammatory responses composed of severe placental macrophage and neutrophil inﬂux coupled to TNF- α production and preterm birth [15] (Table 1). The family of herpes viruses is associated with TLR9 activation and reports demonstrate that active viral shedding during pregnancy is associated with preterm birth outcomes.