Primary Cultures of Female Swine Genital Epithelial Cells In Vitro: a New Approach for the Study of Hormonal Modulation of Chlamydia Infection

Abstract

Previous studies have demonstrated that female reproductive hormones influence chlamydial infection both in vivo and in vitro. Due to the reduced availability of human genital tissues for research purposes, an alternative hormone-responsive model system was sought to study chlamydial pathogenesis. Mature female swine eliminated from breeding programs were selected as the animals of choice because of the similarity of a sexually transmitted disease syndrome and sequelae in swine to a disease syndrome and sequelae found in humans, because of the near identity of a natural infectious chlamydial isolate from swine to Chlamydia trachomatis serovar D from humans, and because a pig's epithelial cell physiology and the mean length of its estrous cycle are similar to those in humans. Epithelial cells from the cervix, uterus, and horns of the uterus were isolated, cultivated in vitro in Dulbecco's minimum essential medium-Hanks' F-12 (DMEM-F-12) medium with and without exogenous hormone supplementation, and analyzed for Chlamydia suis S-45 infectivity. The distribution of chlamydial inclusions in swine epithelial cells was uneven and was influenced by the genital tract site and hormone status. This study confirmed that, like primary human endometrial epithelial cells, estrogen-dominant swine epithelial cells are more susceptible to chlamydial infection than are progesterone-dominant cells. Further, the more differentiated luminal epithelial cells were more susceptible to infection than were glandular epithelial cells. Interestingly, chlamydial growth in mature luminal epithelia was morphologically more active than in glandular epithelia, where persistent chlamydial forms predominated. Attempts to reprogram epithelial cell physiology and thereby susceptibility to chlamydial infection by reversestage, exogenous hormonal supplementation were unsuccessful. Freshly isolated primary pig epithelial cells frozen at ؊80°C in DMEM-F-12 medium with 10% dimethyl sulfoxide for several weeks can, after thawing, reform characteristic polarized monolayers in 3 to 5 days. Thus, primary swine genital epithelia cultured ex vivo appear to be an excellent cell model for dissecting the hormonal modulation of several aspects of chlamydial pathogenesis and infection.

Differences in Chlamydia trachomatis Serovar E Growth Rate in Polarized Endometrial and Endocervical Epithelial Cells Grown in Three-Dimensional Culture

“…Since this finding is quite similar to our previous findings of patchy EB attachment to polarized primary human endometrial gland epithelial cells (33) and to polarized swine genital organ tissue cultured ex vivo (20), in contrast to an even distribution EB attachment pattern in the same cells cultured on glass coverslips, the pattern of attachment appears to be a 3D culture phenomenon, at least in part. The significance of this to receptor complex presentation is not yet clear given the varying reports of EB attachment to nonpolarized epithelia in compartmentalized domains such as caveolae (13,24,35,49), lipid rafts (19,46), and TARP-enriched pedestals (5, 10).…”

“…4E, F, I, and J). This pattern mimics early chlamydial infection previously observed in primary human and pig genital tract epithelial cell models cultured ex vivo (20,33). Inclusions, still somewhat clustered, were easily detected by 24 hpi in the HEC-1B cells (Fig.…”

Comparison of Chlamydia trachomatis serovar L2 growth in polarized genital epithelial cells grown in three-dimensional culture with non-polarized cells

“…It is interesting to note that, firstly, serovar L2 attachment to polarized epithelial cell monolayers on beads appeared evenly distributed (Fig. 1A and D) while serovar E EB attachment/entry was rather patchy and clustered throughout both HEC-1B and HeLa cell monolayers [13], a pattern reminiscent of chlamydial attachment to apical surfaces of polarized primary human and pig genital epithelial cells cultured ex vivo [15,18]. This intriguing difference between strains may possibly be due to the recognition of and binding to different receptor molecules on host cell surfaces that, for serovar E, may be enriched or clustered in some regions or microdomains of polarized epithelial cell apical membranes, such as lipid rafts or caveolae, proposed to be involved in serovar E but not in serovar L2 entry [19,20].…”

Role of Sulfated Glycans in Adherence of the Microsporidian Encephalitozoon intestinalis to Host Cells In Vitro

“…Caco-2 intestinal epithelial host cells were seeded at 10 5 on 8-m porous Transwell inserts (BD Biosciences) in growth medium and maintained for at least 7 days prior to experimentation; 50 million E. intestinalis spores were added to the upper chamber and allowed to adhere to the host cell surfaces for 8 h. The unbound spores were removed by washing, and the host cells were prepared for transmission electron microscopy as previously described (20). Briefly, the host cell-spore sample was fixed in 2% glutaraldehyde-0.5% paraformaldehyde in 0.1 M cacodylate buffer for 2 h at 37°C.…”