No abstract

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent sexually transmitted disease worldwide. Severe clinical sequelae such as pelvic inflammatory disease (PID), tubal occlusion, and tubal infertility are linked to inflammatory processes of chronically infected tissues. The oxygen concentrations in the female urogenital tract are physiologically low and further diminished (0.5–5% O2, hypoxia) during an ongoing inflammation. However, little is known about the effect of a low oxygen environment on genital C. trachomatis infections. In this study, we investigated the host immune responses during reactivation of IFN-γ induced persistent C. trachomatis infection under hypoxia. For this purpose, the activation of the MAP-kinases p44/42 and p38 as well as the induction of the pro-inflammatory cytokines IL-1β, IL-6, IL-8, and MCP-1 were analyzed. Upon hypoxic reactivation of IFN-γ induced persistent C. trachomatis infection, the phosphorylation of the p44/42 but not of the p38 MAP-kinase was significantly diminished compared to IFN-γ induced chlamydial persistence under normoxic condition. In addition, significantly reduced IL-6 and IL-8 mRNA expression levels were observed for reactivated Chlamydiae under hypoxia compared to a persistent chlamydial infection under normoxia. Our findings indicate that hypoxia not only reactivates IFN-γ induced persistent C. trachomatis infections resulting in increased bacterial growth and progeny but also dampens inflammatory host immune signaling responses that are normally observed in a normoxic environment.

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient clearance or persistence of the pathogens may lead to ascending infections of the upper genital tract and are supposed to cause chronic inflammatory damage to infected tissues 1,2 . As a consequence, severe clinical sequelae like pelvic inflammatory disease (PID), tubal occlusion and infertility may occur 3,4 .Most of the research with C. trachomatis has been conducted in epithelial cell lines (e.g. HEp-2 cells and HeLa-229) or in mice. However, as with cell-culture based models, they do neither reflect the physiology of native tissue nor the pathophysiology of C. trachomatis genital tract infections in vivo 5. Further limitations are given by the fact that central signaling cascades (e.g. IFN-γ mediated JAK/STAT signaling pathway) that control intracellular chlamydial growth fundamentally differ between mice and humans 6,7 . We and others therefore established a whole organ fallopian tube model to investigate direct interactions between C. trachomatis and human fallopian tube cells ex vivo 8,9 . For this purpose, human fallopian tubes from women undergoing hysterectomy were collected and infected with C. trachomatis serovar D. Within 24 h post infection, specimen where analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to detect Chlamydia trachomatis mediated epithelial damage as well as C. trachomatis inclusion formation in the fallopian tissue. Video LinkThe 2. Incubate for one hour at 37 °C during constant shaking. 3. Add 20 ml DMEM with 10% FCS and 240 μl Cycloheximide. 4. Incubate in a humidified incubator for 48 h at 37 °C and 5% CO 2 . 5. Add 5 ml sterile glass beads, strong shake to detach and destroy C. trachomatis harboring HeLa-229 cells. 6. Collect supernatant and add another 5 ml of sterile glass beads. 7. Rock three times for 1 min to destroy all HeLa-229 cells and release C. trachomatis. 8. Centrifuge for 5 min at 1000 rpmi and 4 °C to get rid of cellular debris. 9. Collect the supernatant for infection of 8 to 10 175 cm 2 cell culture flasks confluent grown with HeLa 229 cells.10. Incubate every flask with 4 ml SPG buffer and 4 ml supernatant from previous infection for one hour at 37 °C with constant shaking. 11. After one hour add 15 ml DMEM with 10% FCS and 230 μl cycloheximide followed by 48 h incubation at 37 °C and 5% CO 2 . 12. Add 5 ml sterile glass beads, shake to detach and destroy C. trachomatis harboring HeLa-229 cells. 13. Collect the supernatant and add another 5 ml of sterile glass beads. 14. Rock three times for 1 min to destroy all HeLa-229 cells and release C. trachomatis. 15. Centrifuge for 5 min at 1000 rpmi at 4 °C to get rid of cellular debris. 16. Collect the supernatant. 17. Fill 40 ml of supernatant in 50 ml Falcon tubes, centrifuge for 99 min with 11000 rpmi. 18. After centrifugation discard the supernatant and wash the pellet with 1 ml SPG buffer.