Primary ectocervical epithelial cells display lower permissivity to Chlamydia trachomatis than HeLa cells and a globally higher pro-inflammatory profile

Abstract

The tumoral origin and extensive passaging of HeLa cells, a most commonly used cervical epithelial cell line, raise concerns on their suitability to study the cell responses to infection. The present study was designed to isolate primary epithelial cells from human ectocervix explants and characterize their susceptibility to C. trachomatis infection. We achieved a high purity of isolation, assessed by the expression of E-cadherin and cytokeratin 14. The infectious progeny in these primary epithelial cells was lower than in HeLa cells. We showed that the difference in culture medium, and the addition of serum in HeLa cultures, accounted for a large part of these differences. However, all things considered the primary ectocervical epithelial cells remained less permissive than HeLa cells to C. trachomatis serovar L2 or D development. Finally, the basal level of transcription of genes coding for pro-inflammatory cytokines was globally higher in primary epithelial cells than in HeLa cells. Transcription of several pro-inflammatory genes was further induced by infection with C. trachomatis serovar L2 or serovar D. In conclusion, primary epithelial cells have a strong capacity to mount an inflammatory response to Chlamydia infection. Our simplified purification protocol from human explants should facilitate future studies to understand the contribution of this response to limiting the spread of the pathogen to the upper female genital tract.

Metabolic imprint of an intracellular pathogen drives histone hypermethylation and tunes the host transcriptional response to infection

“…To validate these observations in non-cancer derived cells we first used primary epithelial cells derived from the ectocervix of female patients (16). The number of nuclei positive for the three histone methylation marks tested (H3K9me3, H3K27me3, H4K20me3) was significantly higher in the infected cells compared to non-infected cells (Fig.S1C).…”

“…Approval and authorization of the National Data Protection authority (‘Commission Nationale de l’Informatique et des Libertés’, CNIL) have been obtained for the research protocol, in compliance with the Helsinki principles. Primary cells were cultivated in keratinocyte serum free medium (KSFM, Thermo Fisher Scientific) containing 50 mg/L of bovine pituitary extract (Thermo Fisher Scientific) and 5 μg/L of epidermal growth factor (EGF) human recombinant (Thermo Fisher Scientific) (16). They were used between passage 3 and 6.…”

Chlamydia-driven ISG15 expression dampens the immune response of epithelial cells independently of ISGylation

“…The cells were grown and propagated in Gibco ® Dulbecco’s Modified Eagle Medium (DMEM) with GlutaMAX (Thermo Fisher Scientific) and 10% of fetal calf serum (FCS) at 37 °C with 5% of CO 2 . Primary ecto-cervical epithelial cells were isolated as described previously(22) and cultured in Gibco® keratinocyte serum-free medium (K-SFM) supplemented with recombinant human EGF and bovine pituitary extract (Thermo Fisher Scientific, #17005075). The cells were propagated in 75cm 2 flasks (Falcon) and seeded in culture dish (0.1×10 6 cell/well for 24-well plates or 0.2×10 6 cell/well for 12-well plates) 24 h before infection or treatment.…”

Chlamydia trachomatis development requires both host glycolysis and oxidative phosphorylation but has only minor effects on these pathways

“…Approval and authorization of the National Data Protection authority ('Commission Nationale de l'Informatique et des Libertés', CNIL) have been obtained for the research protocol, in compliance with the Helsinki principles. Primary cells were cultivated in keratinocyte serum free medium (KSFM, Thermo Fisher Scientific) containing 50 mg/L of bovine pituitary extract (Thermo Fisher Scientific) and 5 g/L of epidermal growth factor (EGF) human recombinant (Thermo Fisher Scientific) (50). They were used between passage 3 and 6.…”