INITIAL LOCAL CYTOKINE RESPONSES AGAINST NEISSERIA GONORRHOEAE INFECTIONS IN THE HUMAN CERVIX

Abstract

Neisseria gonorrhoeae (GC) is a human-specific pathogen that causes gonorrhea, a common sexually transmitted infection. In women, GC initiate infection by colonizing the cervix. Although GC colonization can cause cervicitis, most female infections are asymptomatic. Asymptomatic colonization of the cervix increases the risk of transmission and progression to severe complications, including pelvic inflammatory disease and infertility. Despite its clinical significance, the mechanisms underlying GC asymptomatic colonization remain unclear. Using a human cervical tissue explant model, which can mimic GC infection in vivo, my Ph.D. research examined the early local cytokine responses to GC cervical colonization, a determining factor for asymptomatic and symptomatic clinical outcomes. Luminex and spatial transcriptomic analyses found that cervical tissue explants constitutively secrete and express a broad spectrum of cytokines, with particularly high levels of the IL-1 receptor antagonist IL-1RA, the anti-inflammatory cytokines IL-10, and the multi-functional cytokine IL-6. During the first 24-h inoculation, GC strain expressing an opacity-associated protein binding to the host receptor CEACAMs (MS11OpaCEA) increased the secretion and transcript levels of both pro-inflammatory, like IL-1α/β, and the anti-inflammatory cytokine IL-10, as well as multi-functional cytokines, like IL-6 and CFS3, but MS11 lacking Opa (MS11∆Opa) induced much less. Notable, the cervix secreted IL-1RA at 100-fold higher levels than IL-1α/β. Cervical secreting levels of soluble IL-6 receptors, required for activating IL-6 inflammatory functions, were 10,000-fold less than IL-6. These results support an anti-inflammatory-dominated cytokine environment of the human cervix, and GC further push it in the anti-inflammatory direction. Using isogenic GC strains and inhibitors, the mechanism underlying GC cytokine induction and the impact of GC-induced cytokines on GC infection were examined. My research found that GC-induced inflammatory cytokine production involved NF-κB activation in both epithelial and subepithelial cells. GC-induced IL-10 production depended on the activation of CEACAM-downstream signaling molecule SHP1/2. Reductions in inflammatory cytokines, TNF-α and IL-1β, by an NF-κB inhibitor did not significantly affect GC colonization, epithelial cell-cell junctions, or epithelial shedding. In contrast, neutralizing IL-10 or blocking its receptor reduced GC colonization and increased ectocervical epithelial shedding and disassembly of epithelial cell-cell junctions. Thesis results suggest that IL-10 plays critical roles in strengthening the cervical epithelium and suppressing the epithelial cell-cell junction disrupting function of inflammatory cytokines, and that GC further elevate the local IL-10 level to prevent bacteria from shedding off with epithelial cells, enhancing colonization.Immunofluorescence and spatial transcriptomic approaches were utilized to identify the types of cervical cells contributing to the local cytokine response to GC infection. Cervical epithelial cells and macrophages are two of the major contributors. IL-1RA protein and mRNA were primarily detected at the ectocervical epithelium. IL-6 protein and mRNA were also detected in ectocervical epithelial cells. MS11OpaCEA colonization increased IL-1RA transcript levels, while MS11ΔOpa switched ectocervical epithelial cells from IL-1RA- to IL-8/IL-6-expressing. GC inoculation did not alter the transcriptomic program of CD68+ macrophages adjacent to the ectocervical epithelium, maintaining the tissue-repair signature. However, GC changed the transcriptomic profiles of macrophages at the explant tissue side, exposed to media and inoculated GC, leading to increased expression of either inflammatory M1- or anti-inflammatory M2 signature genes. These results suggest that the human cervix utilizes high levels of epithelial-secreted IL-RA, low levels of soluble IL-6 receptor release, and tissue-repairing macrophages at the subepithelium to control inflammation induced by colonizing GC when the epithelium prevents GC from entering the tissue. Overall, my research results suggest that GC exploit the local cytokine response of the human cervix, dominant by anti-inflammatory IL-1RA, IL-10, and IL-6, to facilitate colonization and desensitize immune detection, promoting asymptomatic colonization.