Fig. 2.

Cell death induced by seasonal NC/99 IAV depends on viral RNA replication. a Cytokine and chemokine release 4 h after DC infection with either mock, seasonal (NC/99; Tx/91), or pandemic (Cal/09) strains. Supernatants were collected, and cytokine and chemokine levels were measured by multiplex ELISA. The levels of IL-1a, IL-1b, TRAIL, and IL-6 are near or below the limits of detection for this assay. b Transwell experiment (see inset diagram) where mock-infected DC (lower chamber) were exposed to paracrine signaling from NC/99- or mock-infected DC (upper chamber). NC/99-infected cells in the lower chamber were used as a positive control. c Effect of a 30-min pretreatment with actinomycin D (2 μM) on virus-induced cell death. d Effect of reduced infectivity via UV irradiation (NC/99 UV) on virus-induced cell death. The inset shows the percentage of NP-positive cells under the different conditions. b, c, d DC were infected for 8 h with either mock or NC/99 IAV. Percentage of cell death was determined by imaging flow cytometry. Data are representative of two a, d or three b, c independent experiments using DC derived from different donors. n = 3 for experimental groups and for mock-infected controls. ***p < 0.001, ANOVA followed by Tukey’s HSD test. Values shown are median ± s.e.m