Increased TLR-4 expression in murine placentas after oral periodontopathogenic infection

Objectives: In humans the association between maternal periodontitis and prematurity is linked with the systemic hematogenous dissemination of periodontopathogens that reach the uterus and inflame the fetoplacental unit. The objective of this study was to investigate the placental expression of Toll-like (TLR-4) receptors resulting from the inflammatory response to Campylobacter rectus and Porphyromonas gingivalis in a murine model of oral infection. Methods: 40 female Balb/C mice were pretreated for 4 days with Kanamycin/Ampicillin (50mg/kg-25mg/kg) and then randomly assigned to experimental groups: Group 1 (n=12) was infected by oral application of 100µLx10⁹ C.rectus 314/carboxymethylcellulose (Cr+CMC). Group 2 (n=12) received C.rectus plus 100µLx10⁹ P.gingivalis A7436/CMC (Cr+Pg+CMC). Group 3 (n=8) received vehicle (100µL/CMC) and Group 4 (n=8) served as a control. All groups were given ad libitum soft chow enriched with a dextrose solution (30%) as plaque-promoting diet. Mice were infected daily over a 16-week period and mated once/week. Pregnant mice were sacrificed at embryonic day (E)16.5, placental tissues were collected and analyzed for TLR-4 expression by immunohistochemistry (confocal microscopy) followed by RT-PCR analysis. Results: Group 2 (Cr+Pg+CMC) had the lowest fecundity rate (16.7%, HR 0.19[0.041-0.856], P=0.03, Kaplan-Meier) compared to Group 1 (Cr+CMC, 58.3%, HR 0.87[0.310-2.154], P=0.68) and to Groups 3 and 4 (75% combined). After normalization, TLR-4 expression was upregulated in Group 1 (1.98±0.886 fold difference, P<0.01 ANOVA) and Group 2 (1.29±0.871) compared to controls. Immunohistochemistry of placentas from Groups 1 and 2 showed increased expression of TLR-4 in trophoblasts, mainly in the labyrinth facing maternal blood spirals. Conclusions: Significant TLR-4 upregulation was observed in placentas after oral infection by C.rectus and C.rectus/P.gingivalis combined infection. The TLR-4 pathway is a key regulator during labor initiation, so the abnormal upregulation of this pathway may explain in part how maternal periodontitis and periodontopathogenic bacteria could be linked to prematurity in pregnant women.