Persistent Mycoplasma genitalium infection of human endocervical epithelial cells elicits chronic inflammatory cytokine secretion

Abstract

Infection with Mycoplasma genitalium has been associated with male and female urogenital disease syndromes, including urethritis, cervicitis, pelvic inflammatory disease (PID), and tubal factor infertility. Basic investigations of mucosal cytotoxicity, microbial persistence, and host immune responses are imperative to understanding these inflammatory urogenital syndromes, particularly in females, considering the potential severity of upper tract infections. Here, we report that M. genitalium can establish long-term infection of human endocervical epithelial cells that results in chronic inflammatory cytokine secretion and increased responsiveness to secondary Toll-like receptor (TLR) stimulation. Using a novel quantitative PCR assay, M. genitalium was shown to replicate from 0 to 80 days postinoculation (p.i.), during which at most time points the median ratio of M. genitalium organisms to host cells was ≤10, indicating that low organism burdens are capable of eliciting chronic inflammation in endocervical epithelial cells. This observation is consistent with clinical findings in women. Persistently secreted cytokines predominately consisted of potent chemotactic and/or activating factors for phagocytes, including interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein 1β (MIP-1β). Despite persistent cytokine elaboration, no host cell cytotoxicity was observed except with superphysiologic loads of M. genitalium, suggesting that persistent infection occurs with minimal direct damage to the epithelium. However, it is hypothesized that chronic chemokine secretion with leukocyte trafficking to the epithelium could lead to significant inflammatory sequelae. Therefore, persistent M. genitalium infection could have important consequences for acquisition and/or pathogenesis of other sexually transmitted infections (STIs) and perhaps explain the positive associations between this organism and human immunodeficiency virus (HIV) shedding.

Fig 1

Microscopic cytotoxicity in response to M. genitalium G37 infection of human endocervical epithelial cells. Immortalized human endocervical epithelial cell lines (A2EN and ShEN101), which were derived from two different donors, were inoculated with escalating doses of M. genitalium (MOIs of 2 to 200) and then observed via light microscopy for gross signs of monolayer disruption. Inoculation of 200 organisms per cell resulted in rounding of A2EN and ShEN101 cells by 2 days (d) p.i. at the highest inoculum (MOI of 200). Continued deterioration of the cell monolayer occurred between 4 and 14 days p.i. when significant detachment of cells was observed at the highest inoculum (MOI of 200). No obvious microscopic signs of monolayer disruption were observed at an MOI of 20 or at an MOI of 2 (data not shown) compared to mock-infected cells at any time point from 2 to 14 days p.i. Findings from the low-passage-number ShEN101 cells were indistinguishable from A2EN and omitted for the sake of simplicity.

Fig 2

Acute cytotoxicity of M. genitalium to endocervical epithelial cells A2EN (A) and ShEN101 (B) was dose dependent. Human endocervical epithelial cells were exposed to various inocula of M. genitalium G37 (black fill) or an equal volume of culture medium as a negative control (no fill). After 24 or 48 h of infection, LDH was measured from culture supernatants as a marker for epithelial cell lysis. Heat-inactivated organisms (gray fill) served as controls to determine whether viability was necessary for immune activation. A statistically significant LDH increase relative to controls is noted by an asterisk at a P value of <0.05 (ANOVA).

Fig 3

Replication of M. genitalium in human endocervical epithelial cell cultures. (A) After inoculation of A2EN or ShEN101 cells with M. genitalium G37 (MOI of 10), bacterial replication was monitored by a qPCR assay, as described in Materials and Methods, from 0 to 80 days p.i. (B) Differences in qPCR titers are expressed as the mean ratio (±standard error of the mean) of the cellular to supernatant fraction at each time point from three independent studies.

Fig 4

Azithromycin but not gentamicin treatment ablated endocervical cytokine responses from persistently infected cells. In order to determine whether killing of M. genitalium would eliminate the observed inflammatory response, persistently infected A2EN cells (36 days p.i.) were exposed to either 200 μg/ml of gentamicin (Gm) for 2 h to kill extracellular organisms or 8 μg/ml of azithromycin (Az) for 2 days to kill both intra- and extracellular organisms. As controls, antibiotic-treated mock-inoculated cells were processed in parallel. IL-8 secretion was measured from culture supernatants 2 (A) and 14 days (B) after inoculation. Data are presented as pg/ml of secreted IL-8 ± standard errors of the means. As determined using a Student t test, significant differences versus appropriate mock-inoculated controls are noted as follows: *, P < 0.05; **, P < 0.005.

Fig 5

Endocervical epithelial cytotoxicity occurs only with high M. genitalium loads. In three independent replicate experiments, we tested the ability of M. genitalium to elicit epithelial cell toxicity as measured by LDH secretion at 14 and 36 days after inoculation. Marked differences in cell numbers and morphologically abnormal cells prevented accurate comparisons of LDH secretion after 60 days p.i. As a positive control and for calculation of percent cytotoxicity, an equal number of cells were detergent lysed to determine the maximum releasable LDH.