Fig 4. C4BP is necessary and sufficient for serum-mediated suppression of neutrophil anti-gonococcal activity.

(A-C) Adherent, IL-8-treated neutrophils were exposed to OpaD+ Gc incubated in PBS+ (red) or the following (all at 25% final concentration) as in Fig 1A: (A) heat inactivated serum that was C4BP-replete (brown; Lund University) or C4BP-depleted (depl, blue); (B) C4BP-replete serum (orange; Lund Universtiy) or 50 μg/mL purified C4BP (purple); (C) C4BP at the indicated concentrations. In (A-B), two-way ANOVA with Sidak’s post-hoc test was used to compare each condition within each time point for 3 independent experiments; (C) used one-way ANOVA followed by Tukey’s post-hoc comparisons to compare each condition to PBS alone (0 μg/mL C4BP). (D-E) Neutrophil ROS production was measured as in Fig 1C in response to OpaD+ Gc that was incubated (D) in PBS, heat-inactivated C4BP-replete serum (brown; Lund University), heat inactivated C4BP-depleted serum (blue), or purified C4BP at 50 μg/mL (purple); or (E) in the indicated concentration of C4BP. (F) Neutrophils were exposed to OpaD_porS-23 Gc or OpaD_porKan Gc, with or without C4BP addition to the infection milieu (50 μg/mL), and bacterial survival after 60 minutes was measured as in (A). Results are the mean ± SEM of 3 independent experiments; two-way ANOVA with Sidak’s post-hoc comparisons were used to compare each condition. *p<0.05, **p<0.01,****p<0.0001. (G) OpaD+ Gc (grey solid lines), OpaD_porS-23 Gc (black dotted lines), and OpaD_porKan Gc (purple solid lines) alone (PBS; triangles), incubated with C4BP (open circles), or added to wells containing C4BP at a final concentration of 50 ug/mL (C4BP “in trans”; stars) were exposed to neutrophils, and ROS production was measured as in (E). Results in (D, E, and G) are one representative of 3 independent experiments.