Characterization of the invasive and inflammatory traits of oral Campylobacter rectus in a murine model of fetoplacental growth restriction and in trophoblast cultures

Abstract

Campylobacter species (C. jejuni, C. fetus) are enteric abortifacient bacteria in humans and ungulates. Campylobacter rectus is a periodontal pathogen associated with human fetal exposure and adverse pregnancy outcomes including preterm delivery. Experiments in pregnant mice have demonstrated that C. rectus can translocate from a distant site of infection to the placenta to induce fetal growth restriction and impair placental development. However, placental tissues from human, small-for-gestational age deliveries have not been reported to harbor C. rectus despite evidence of maternal infection and fetal exposure by fetal IgM response. This investigation examined the temporal relationship between the placental translocation of C. rectus and the effects on fetal growth in mice. BALB/c mice were infected at gestational day E7.5 to examine placental translocation of C. rectus by immunohistology. C. rectus significantly decreased fetoplacental weight at E14.5 and at E16.5. C. rectus was detected in 63% of placentas at E14.5, but not at E16.5. In in vitro trophoblast invasion assays, C. rectus was able to effectively invade human trophoblasts (BeWo) but not murine trophoblasts (SM9-1), and showed a trend for more invasiveness than C. jejuni. C. rectus challenge significantly upregulated both mRNA and protein levels of IL-6 and TNFalpha in a dose-dependent manner in human trophoblasts, but did not increase cytokine expression in murine cells, suggesting a correlation between invasion and cytokine activation. In conclusion, the trophoblast-invasive trait of C. rectus that appears limited to human trophoblasts may play a role in facilitating bacterial translocation and placental inflammation during early gestation.

Fig. 1

C. rectus invasiveness in murine placentas and mammalian trophoblasts. Immunofluorescence analysis of murine placentas: (a–c) representative images in murine placentas from non-infected (a) and infected mice (b and c). C. rectus was seen in placentas of infected mice (64%) but not in the placentas of control mice. The presence of bacterial cells was rather scarce and was not observed in the majority of the scanned fields: (d–i) representative images from in vitro trophoblastic cell infection experiments and non-infected controls. Bacteria were found extracellularly attached to murine trophoblasts, or present in the cell junctions within cells (e) without apparent evidence of cytoplasmic invasion (f). Conversely, C. rectus and C. jejuni were detected intracellularly in the human trophoblast BeWo cells (h and i). (j–l) The canine epithelial MDCK cells served as positive control target cells using a highly invasive intestinal pathogenic strain of C. jejuni. For all image stacks, red stain corresponds to F-actin stained with Texas Red-conjugated Phalloidin. Green-yellow fluorescent cells represent bacteria stained with an FITC-conjugated Campylobacter-specific antibody. Magnifications: [a–c] at 63× na 1.4 plan apo and [d–l] at 100× na 1.3 plan neo fluar lenses. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 2

Proinflammatory cytokine expression in human trophoblasts after in vitro C. rectus infection. Box plots depict mean ± SD cytokine concentration values. Cell supernatants were harvested and cytokines were quantified by ×MAP multiplexing. Human IL-6 and TNFα were significantly increased in a MOI-dependent manner. *P < 0.05 and **P < 0.0001.