Sexually transmitted diseases (STDs) present a major global health problem in the female population of reproductive age. Infections caused by Chlamydia trachomatis (CT) and Herpes Simplex Virus (HSV) are among the leading STDs reported in the United States and throughout the world. Clinical reports have indicated that co-infections between sexually transmitted agents such as Chlamydia trachomatis and Herpes Simplex Virus-2 (HSV-2) increase the pathogenesis and resulting sequelae produced by these microorganisms. The following studies were designed to elucidate the Toll-like receptors (TLR) that identify and bind the pattern recognition molecules of CT and HSV, the pathways activated upon binding, and to evaluate the cytokine responses of female reproductive tract epithelial cells to single and co-infection by these agents. Immortalized primary genital epithelial cells and primary genital tract epithelial cells were infected with CTD, CTE or HSV and treated with supernatants from previous infections by CTD and CTE and HSV. Cell culture fluids and cell pellets were collected and analyzed for cytokine production, TLR RNA expression, percent infectivity, and expression of phosphorylated protein involved in the TLR pathway.
Analysis of the cytokine profile in various epithelial cells indicated both CT and HSV affected the epithelial cell cytokine response. A trend was observed that the lower tract epithelial cells often induced up-regulation of several cytokines while the upper tract epithelial cells induced up-regulation of fewer cytokines and down-regulated several cytokines as well. This observation indicates a possible higher or more intense immune response at the lower regions of the female reproductive tract. As the vaginal vault epithelial cells are the first cells come in contact with an invading pathogen, the ability to activate an intense immune response would benefit the host by quickly initiating pathways to begin defensive mechanisms and prevent the spread of the pathogen. These protective mechanisms would defend the female reproductive tract against ascending infection where the damage to the epithelial tissue is the very detrimental to reproductive function, such as scarring. In addition, the upper epithelial cells may not respond as intensely as the lower tract due in order to prevent the activation of an uncontrolled cytokine response, as problems that arise from a rampant inflammatory response include tubal scaring or pelvic inflammatory disease.
Based on available data, our findings support previous research on the activation of the various TLRs upon infection with CT or HSV. However, our study further explored the role of TLRs in the activation of signaling cascades in the presence of supernatant from previous infections. In addition to published findings, our data suggests that HSV may also utilize the TLR4 as well as the TLR2, TLR3 and TLR9 pathway, primarily in the vaginal epithelium. We also confirmed that CT infected cells do not use the TLR4 pathway. However we did not observe a significant increase in TLR2 as indicated by other studies. Our findings, additionally, are novel in that this is the first known report of the effect of co-infections on the expression of TLRs in the female reproductive tract. TLR2 RNA expression was increased or up-regulated in the co-infection models, indicating that these cells may activate a more severe or intense immune response at when single infections alone occur. A more intense immune response may result in the activation of an uncontrollable inflammatory response, through the recruitment of NK cells, macrophages, T and B cells and other immune modulators to the site of infection. The consequence of such an intense immune response may include reproductive tract damage and scarring, which has been reported in the clinical literature for patients with multiple sexually transmitted infections.
The western blot analysis of the activation of downstream proteins in the MyD88 dependent and independent pathways was performed to elicit which pathway was activated during a co-infection by Chlamydia trachomatis and HSV-2 and determine if the pathway was different from the one activated by single infections. The data showed that the upper tract cells upon co-infection down-regulated the phosphorylation in both pathways, which could potentially result in a suppressed immune response to the co-infecting pathogens. The lower tract epithelial cells upon co-infection increased the phosphorylated protein expression, indicating the activation of the MyD88 dependent pathway and infection in this region may result in a severe inflammatory response. Our data showed that co-infection by CT and HSV in epithelial cells treated with supernatants from previous infection results in the activation of pathways that differ among the four reproductive epithelial regions, vaginal, ectocervical, endocervical, and fallopian tube. While the expression of proteins were not significantly changed, the pattern indicated that the presence of culture fluids from a previous infection drives the cells to activation the pathways common for that pathogen, even when a secondary pathogen would normally activate a different pathway as a single infection. (Abstract shortened by UMI.)