In the current study, we report an experimental in vitro examination of the role of gonococcal pili during epithelial invasion and cytokine production at the initial stages of infection of the human Fallopian tube. Experimental infection based on cell lines and cultures has provided a theoretical model for gonococcal infection in which pili have a prominent role in the initial attachment to the host cell [18
]. We observed in our study that the presence of pili enhanced gonococcal adherence to the tubal epithelium within 3
h of infection compared with the corresponding nonpiliated variant but thereafter did not appear to offer a significant advantage to gonococci to colonize the epithelial cell surface. We have shown previously that a clinical Pil−
gonococcal strain attached and invaded the epithelium of tubal explants [17
] and the appearance of Pil−
bacteria during experimental infection has also been reported [28
]. In our study, pilus expression also did not confer any advantage to the gonococcus for invasion. Moreover, because the strains we used in our experiments were both Opa negative, our data also suggested an Opa-independent mechanism for invasion of the tubal epithelium. The redundancy of Opa is consistent with reports demonstrating that Opa−
variants were as infective as Opa+
gonococci in human experimental infections [30
], and adhesion and phagocytosis of Pil−
gonococcal strains by human dendritic cells were influenced by differences in their lipooligosaccharides [31
Taken together with the reported literature, our findings therefore suggest that mechanisms other than pilus or Opa-mediated binding to surface molecules of the epithelial cells operate with high efficiency during gonococci invasion of the Fallopian tube. The exact nature of these mechanisms is unclear, although it has been reported, using recombinant E. coli
expressing specific gonococcal components, that invasion of FT epithelia depended on the expression of combinations of Opa, porin, and lipooligosaccharide [32
]. However, it is also likely that the endocytic properties of the oviductal epithelium [33
] probably contribute to uptake of gonococci.
In the current study, expression of pili appeared to have a role in the regulation of cytokine responses following gonococcal infection. Although both Opa−
gonococcal strains induced secretion of IL-1β
, GM-CSF, MCP-1, and MIP-1β
, the stimulatory effects were significantly higher with the Pil−
bacteria compared to Pil+
bacteria. Thus, expression of gonococcal pili appeared to attenuate cytokine-chemokine responses in the mucosal epithelial cells of the human Fallopian tube, although the contribution of other uncharacterized genetic differences cannot be categorically ruled out. Or, alternatively, the absence of the large numbers of surface-located pili structures results in remodeling of the OM of the gonococcus to allow increased interactions with the epithelial cell LOS receptors, leading to increased cytokine production. These findings are in contrast to other studies that have investigated the involvement of pilus expression in determining the magnitude of the cytokine response. For example, during infection of a human monocytic cell line with Salmonella typhi
bacteria induced higher levels of IL-6 production and NF-κ
B activation than nonpiliated bacteria [34
]. However, we should stress that the cytokine pattern observed in our experiments might not necessarily represent the in situ infection, because in vitro mucosal explants are devoid of blood and/or tissue-associated immune effector cells [35
], whose contribution to the local cytokine pool is to be expected during the advanced stages of salpingitis when bacteria reach deep into the subepithelial tissue.
By contrast to pilus expression, the presence of Opa protein was not a contributory factor to cytokine attenuation in the FT epithelium itself. However, it has been shown with human dendritic cells that Opa proteins can modulate innate and adaptive immune responses, by directly suppressing T-cell receptor signaling through engagement of coinhibitory CD66a/CEACAM1 receptor, thereby raising the threshold for CD4+
T cell activation and proliferation to activating stimuli [36
] and interfering with antibody production by promoting human B-cell death [38
]. Thus, Opa expression may play an indirect role in inhibiting innate recognition during FT infection by targeting the function of patrolling submucosal dendritic cells.
In summary, our data suggest a modulatory role for gonococcal pili during infection of the human tubal mucosa. Expression of pilus enhances the initial rapid attachment of the gonococcus to the Fallopian tube mucosal epithelium but significantly attenuates the local production of several inflammatory mediators. Although phase variation of pilus and opa expression in the upper reproductive tract is likely to be a dynamic process during interactions with the host, it is likely that the Pil− phenotype is probably present at a lower percentage of the gonococcal population. Switching off pilus expression would not be beneficial to the gonococcus in that an excess inflammatory response contributory to LOS-mediated stimulation could result. Thus, a consequence of limiting the innate response of the epithelium is to reduce immune effector cell recruitment to the site of infection. This would add to the mechanisms that could allow repeated gonococcal infections in the same individual without ever generating protective immunity.