Fig. 5

OMVs induce antiviral signalling in macrophages via TRIF. A THP-1 Dual reporter cells induce the expression of secreted embryonic alkaline phosphatase (SEAP) after activation of the NF-κB pathway and the expression and secretion of Lucia luciferase upon activation of the IRF pathway. Additionally, TRIF^−/− cells have a stable knockout of the adapter molecule TRIF. (B-F) THP-1 reporter cells (= Dual; black bars) and TRIF^−/− cells (grey bars) were differentiated and subsequently stimulated with KpOMV (1 µg/mL) for 20 h or left untreated for control. After the indicated time, supernatant, RNA and/or proteins were collected. B Representative Western Blot image of Mx1 protein expression. C + D Lucia reporter activity (C) and SEAP reporter activity (D) was determined in cell culture supernatant. The same supernatant was used to determine the activity of both reporters. E Relative mRNA expression for STAT- and NF-κB-dependent target genes (from top to bottom: IFIT1, IFI44, Mx1, CXCL8) was determined by qPCR and results are normalized to RPS18 and depicted relative to unstimulated Dual control. Fold changes were log2 transformed. Cells were additionally infected with A/WSN/33(H1N1) (MOI 0.1) for 24 h (F) or VSV (MOI 0.1) for 12 h (G). Viral replication was determined by plaque assay. Bars show mean values of four (C–F) to five (G) independent experiments + SEM. Statistics: 2-way ANOVA (C–G); *p < 0.05, **p < 0.01, ****p < 0.0001; * compared to unstimulated Dual control, # as depicted in the graph; ns = not significant; n = 4–5