Fig 2. DIV crude supernatant induces inflammatory cytokine secretion independent of antibody signaling.

(A) Left: Flow-cytometric histograms of 24-hpi DENV E-protein expression in mobilized monocytes after inoculation with mock supernatant, DIV crude supernatant alone, or DIV crude supernatant that was pre-incubated with 0.1 μg/ml mAb 2D22 (enhancing), 2 μg/ml mAb 2D22 (neutralizing), or 2 μg/ml mAb 1C17 (enhancing). Right: Measurement of secreted IL-1β at 24 hpi for corresponding samples. (B–F) Mobilized monocytes were inoculated with mock medium, DIV crude supernatant alone, or DIV crude supernatant that had been incubated with 0.1 μg/ml of mAbs 5G22, 1F4, 2D22, or 1C17. At 1 hpi, cells were washed to remove inoculum and resuspended in fresh medium. At 24 hpi, supernatants were collected, and cytokine secretion was evaluated by multiplex array, including inflammatory cytokines IL-1β (B), TNF (C), IL-12 (D), MIP-1α (E), and MIP-1β (F). Dashed lines indicate mean concentration induced by mock medium, except for panel D, in which mock-induced values fell below the lower limit of detection. (G) Measurement of intracellular DENV E-protein at 24 hpi in mobilized monocytes that were incubated with PBS or Fc-receptor binding inhibitor prior to inoculation with DIV crude supernatant. (H) Secreted IL-1β at 24 hpi using supernatants from 2G. Tests used: One-Way ANOVA (within DENV treatment) with Tukey’s post-test (A), One-Way ANOVA with Dunnett’s post-test (B–F), and Two-Way ANOVA with Bonferroni’s post-test (G and H).