Fig 1. Incubation with normal human serum decreases neutrophil anti-gonococcal activity.

(A) Adherent, IL-8-treated primary human neutrophils were exposed to Opaless Gc, OpaD+ Gc, or OpaD+ Gc that were preincubated in 25% normal human serum from healthy subjects (UVA). CFU were enumerated from neutrophil lysates at 0, 30, and 60 minutes (min) post infection, and bacterial survival is presented as the mean ± SEM of CFU at the indicated time point relative to the mean CFU for the same condition at 0 minutes for 8 independent experiments. Two-way ANOVA with Sidak’s post-hoc comparisons were used to compare each condition within each time point. In (B), OpaD+ Gc was mixed with the indicated percentage of normal human serum (UVA), and survival from neutrophils at 60 minutes was performed as in (A). One-way ANOVA followed by Tukey’s post-hoc comparisons was used to compare each condition to PBS alone (0% serum). *p<0.05, **p<0.01,***p<0.001. (C) OpaD+ Gc alone (PBS) or incubated in the indicated percentage of normal human serum (UVA) was exposed to neutrophils in suspension at an MOI of 100 in the presence of luminol. ROS production was measured over the course of 60 minutes (min) as the relative light units (RLU) generated by luminol-dependent chemiluminescence. (D) ROS production was measured as in (C) from OpaD+ Gc incubated in serum (UVA), complement component 3-depleted serum (“C3-depl serum”), or heat-inactivated serum (“HI serum” (56°C, 30 minutes)). (C-D) Results are one representative of 3 independent experiments.