Abstract
The Gram-negative diplococcus Neisseria gonorrhoeae causes the prevalent sexually transmitted infection, gonorrhea. Gonorrhea is a major global health concern, with over 78 million infections world-wide per year. Of particular concern are the lack of a lasting immune response, the lack of a vaccine, and the rise in antimicrobial resistance, with some strains resistant to multiple antibiotics. Female infection is often asymptomatic, and as such, women are particularly at risk for severe clinical consequences including pelvic inflammatory disease, ectopic pregnancy, and infertility.
Infection with the human-specific pathogen Neisseria gonorrhoeae (Gc) is characterized by robust neutrophil influx that is insufficient to clear Gc. While infected men are typically symptomatic and seek treatment, female infection is often asymptomatic. Importantly, women have neutrophilic cervicitis whether or not they notice symptoms. Sustained infection and neutrophilic inflammation contribute to host cell damage and serious clinical sequelae in women including pelvic inflammatory disease and infertility. While previous work has shown that Gc interacts with epithelial cells through various surface-exposed features, the specific Gc-cervical cell interactions that stimulate neutrophil transepithelial migration were unknown. Previous models used to study Gc infection do not incorporate neutrophils and do not completely mimic initial infection and neutrophil influx at the cervix. Therefore, to investigate this site-specific neutrophilic inflammation, we established a three-component gonococcal-endocervical epithelial cell-neutrophil co-culture system to investigate neutrophil influx following infection. Using this system, we find that polarized End1 monolayers support apical infection with Gc and that primary human neutrophils migrate from the basal-to-apical direction following apical infection. Neutrophil transepithelial migration required both an epithelial cell contribution and bacterial-epithelial cell contact.
Bioactive lipids derived from arachidonic acid (AA) by lipoxygenases include leukotriene B4 (LTB4) and hepoxilin A3 (HXA3) and are potent neutrophil chemoattractants. Chemical inhibition of epithelial PKC and cPLA2 that lead to AA release inhibited neutrophil transepithelial migration to Gc. Neutrophil transepithelial migration did not require epithelial 5-lipoxygenase activity but was amplified by neutrophil 5-lipoxygenase-dependent production of LTB4. Pharmacologic and genetic targeting of endocervical 12-lipoxygenase activity inhibited neutrophil transepithelial migration to Gc. Additionally, neutrophil transepithelial migration was abrogated by selective inhibition of MRP2 channels that transport HXA3 and by addition of recombinant human soluble epoxide hydrolase (sEH) that specifically degrades HXA3. Further, trypsin-shaved Gc stimulated significantly less neutrophil transepithelial migration. Our work supports a model in which Gc-endocervical cell contact stimulates epithelial signaling events leading to 12-lipoxygenase activation, HXA3 production, and consequent neutrophil transepithelial migration that is amplified by neutrophil-derived LTB4. These studies are the first to define the molecular events that initiate and drive neutrophilic inflammation in the context of physiologically relevant cervical infection by Gc. This work reveals novel adjunctive therapeutic targets for limiting the deleterious sequelae associated with neutrophilic inflammation to gonorrhea in women.
