Viral infection of the pregnant cervix predisposes to ascending bacterial infection

Abstract

Preterm birth is the major cause of neonatal mortality and morbidity, and bacterial infections that ascend from the lower female reproductive tract are the most common route of uterine infection leading to preterm birth. The uterus and growing fetus are protected from ascending infection by the cervix, which controls and limits microbial access by the production of mucus, cytokines, and antimicrobial peptides. If this barrier is compromised, bacteria may enter the uterine cavity, leading to preterm birth. Using a mouse model, we demonstrate, to our knowledge for the first time, that viral infection of the cervix during pregnancy reduces the capacity of the female reproductive tract to prevent bacterial infection of the uterus. This is due to differences in susceptibility of the cervix to infection by virus during pregnancy and the associated changes in TLR and antimicrobial peptide expression and function. We suggest that preterm labor is a polymicrobial disease, which requires a multifactorial approach for its prevention and treatment.

Figure 1. Murine model of ascending bacterial infection

Non-pregnant (A) and pregnant (B) mice were visualized 24 hours after receiving intravaginal injections of E. coli labeled with RFP. Legend indicates that red color in non-pregnant animal has ascending bacterial infection in utero. NT=no bacterial injection, Bacteria=intravaginal bacterial injection; n=6. (C) Time course of murine model of ascending bacterial infection. Non-pregnant mice were visualized 12, 24, 48 and 72 hours after receiving intravaginal injections of E. coli labeled with GFP. Bacteria are visualized in the vagina and cervix at 12h, found throughout the reproductive tract at 24 and 48 hr, and are diminishing 72 hr post-infection.

Figure 2. Ureaplasma urealyticum ascends into the uterus in MHV68 infected animals

Mice were infected with MHV68 or DMEM (control) at E8.5 of pregnancy, received an intravaginal injection of pathogenic U. urealyticum at E15.5, and were sacrificed (Sac) at E16.5 (A). NT= mice with no MHV68, MHV68= virus treated animals. Both groups received U. urealyticum; decidua and lymphoid aggregates are represented as fold difference from the same tissue from mice with no viral infection. Mice with MHV68 had higher concentrations of bacteria in the decidua and lymphoid aggregates (Lymph Agg) compared to mice without viral infection (B). n=5

Figure 3. MHV68 infection decreases TLR expression in the pregnant cervix

NT= no MHV68, MHV68= mice infected with virus. TLR mRNA expression was higher or did not change in the cervix of non pregnant MHV68 infected animals relative to non infected controls (A) but specific TLR mRNA was significantly decreased in the cervix of MHV68-infected pregnant animals (B). Significance as denoted by bars is a p <.05. n=5

Figure 4. MHV68 infection reduces cytokine response to bacteria in the cervix

Cervical cytokines were measured using luminex technology; “NT” are cytokines from the cervix from animals that did not receive MHV68 or bacteria, “Bacteria” are cytokines from the cervix of animals with an intravaginal bacteria injection, but no MHV68, and “MHV + Bacteria” are cytokines from the cervix of animals that received an intravaginal injection of bacteria while having an MHV68 infection. A number of cytokines and chemokines (A–C) were upregulated in the cervices of animals with artificial intravaginal bacterial infection, while this response was significantly reduced in animals with MHV68 infection. Beta defensin 1 (BD1), beta defensin 3 (BD3), beta defensin 4 (BD4) and beta defensin 14 (BD14) mRNA expression was also reduced in cervices from MHV68 infected animals (D). Significance is denoted by bars and is p <.05. n=5

Figure 5. MHV68 infects the cervix of pregnant mice

Both pregnant and non-pregnant mice had similar systemic infections as determined by MHV68 ORF53 in the spleen (A), but pregnant mice also had high MHV68 titers in the cervix while non pregnant mice had no cervical infection (B). n=4, p<.001

Figure 6. Sex hormones play a role in MHV68 infection of cervix tissue

Mice were overiectomized (OVX) and after 3w, given either P4+E2 or vehicle for 3d; they were then injected with MHV68, and remained on either hormone treatments or vehicle for 7d (A). Seven days after infection, MHV68 was detected in the cervices of mice treated with hormones, but absent in vehicle treated controls (B). n=4, p< .05

Figure 7. Integrins play a role in MHV68 entry into cervical cells

Human ectocervical cells (ECT1) were pretreated with fibronectin to block integrin-ligand interactions and then infected with MHV68; 24h after infection, MHV68 was found to be partially blocked from cells treated with fibronectin (Fibronectin) compared to cells treated with MHV68 but no fibronectin (NT)(3 independent experiments) (A). Western blot analysis showed integrin alpha 3 and beta 1 were upregulated in the pregnant cervix of infected mice (B) n=3. Antibodies blocking integrin alpha 3 (MHV68 + anti-integrin a3 ab) partially blocked MHV68 infection, while treatment with non specific IgG (MHV68 + IgG) did not block MHV68 infection as compared to cells only treated with MHV68 (MHV68 only) (Representative of 3 independent experiments with a minimum of 3 animals per group) (C). Significance is denoted by bars and is p <.05.

Figure 8. Model of polymicrobial disease during pregnancy

(A) Commensal bacteria are located in the lower reproductive tract, and during healthy pregnancy, the uterine cervix provides protection against bacteria ascending into the upper reproductive tract. If the protection provided by the uterine cervix is jeopardized, bacteria can ascend from the lower tract, to the decidua and amnion, leading to inflammation and pregnancy complications such as preterm birth. We propose a model of polymicrobial disease during pregnancy: in this model, pregnancy and the associated sex hormones increase the susceptibility of the cervix to viral infection. Viral infection then results in a decrease in the protection against ascending bacteria. The decrease in protection can then lead to intrauterine inflammation in response to bacteria and preterm birth (B).