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Fig. S1. Preceding influenza infection leads to elevated levels of IL-6 after co-infection with SP. MDDCs were infected with IAV for 4 h before SP was added. The cells were incubated for a further 18 h before the concentration of IL-6 in the supernatants was measured by ELISA. Different infection conditions were tested for their potential to induce an elevated cytokine response.

A. IAV/heat-inactivated (HI) IAV moi 0.5 and SP moi 1.

B. IAV/HI-IAV moi 0.5 and viable or gentamicin-killed SP moi 1.

C. Increasing moi of IAV and SP moi 1.

D. Increasing moi of SP.

The graphs show cytokine concentrations derived from cells of one representative donor out of (A) six, (B) two, (C) seven, (D) three different donors.

Fig. S2. Clearance of phagocytosed SP after IAV infection and bacterial uptake after infection with Semliki Forest virus (SFV).

A. MDDCs were infected with IAV for 4 h before SP was added. At indicated time points the cells were lysed and viable, intracellular SP was enumerated. The graph shows mean ± SEM of independent experiments with three different donors.

B. MDDCs were infected with SFV at different moi for the indicated time points before bacterial uptake was monitored. Values represent mean ± SEM of independent experiments with a minimum of three different donors. Statistical analysis was performed using paired Student's t-test. (***P < 0.001).

Fig. S3. Increased secretion of IL-6 can be stimulated by a combination of different TLR agonists and SP. Different doses of a TLR3 (A), TLR7/8 (B) or TLR4 agonist (C) were applied for 4 h before SP was added. The cells were incubated for another 18 h and concentration of IL-6 in supernatants was determined by ELISA. The graphs show cytokine concentrations derived from cells of one representative donor out of (A, B) three, (C) two different donors.

Fig. S4. IAV infection does not enhance uptake and digestion of SP. MDDCs were seeded on glass slides and infected with SP only or sequentially infected as described before. The cells were fixed with paraformaldehyde 4 h after addition of SP and stained with specific antibodies for SP and Hoechst DNA stain. Four hundred cells per donor were examined and the percentage of cells with cytoplasmic stain for SP was determined. The numbers show the average frequency for independent experiments with three donors ± SEM. Statistical analysis was performed using paired Student's t-test.

Fig. S5. Supernatants of IAV-infected MDDCs prime non-infected cells to launch a stronger IL-6 response towards SP.

A. MDDCs were infected with IAV or treated with supernatants of mock- (SN mock) or IAV-infected MDDCs (SN IAV) and incubated for 4 h. The cells were then infected with SP and further incubated for 18 h before the concentration of IL-6 in the supernatants was measured by ELISA. The graph shows results of one representative experiment out of five with different donors. No infectious IAV particles in supernatant of infected MDDCs detectable.

B. MDDCs were incubated with supernatants of uninfected (top row), IAV infected (middle) or supernatants of IAV-infected MDDCs which were spiked with IAV stocks (bottom row) and incubated for 16 h. The cells were then fixed with paraformaldehyde and stained for the presence of IAV nucleoprotein.

C. MDDCs were infected with IAV, or incubated with supernatants of uninfected or IAV-infected MDDCs. After 16 h of incubation the cells were lysed and total RNA was isolated and assayed for the presence of IAV M1 mRNA. Ct values were normalized against γ-actin and the relative induction of genes was calculated using the ΔΔCt method.

Values represent mean ± SD of two independent experiments with different donors.

Fig. S6. IL-6 secretion after IFN-priming and infection with SP. MDDCs were incubated with different concentrations of recombinant IFN-α 4 h prior to infection with SP. The cells were further incubated for 18 h before the concentration of IL-6 in the supernatants was measured by ELISA. The graph shows mean ± SEM from three independent experiments with different donors.

Fig. S7. Gö6976 inhibits expression of IL-12p70. MDDCs were infected with IAV for 4 h before SP and Gö6976 were added. The cells were incubated for another 18 h and the concentration of (A) IL-12p70 and (B) IL-12p40 in supernatants were determined by ELISA. Values represent mean ± SEM of three independent experiments with different donors.

Fig. S8. mRNA induction kinetics of IFN-β and IFN-α2. MDDCs were infected with IAV for 4 h before SP was added. The cells were then incubated and lysed at the indicated time points. Total RNA was isolated and assayed for the presence of (A) IFN-β and (B) IFN-α2 mRNA. Ct values were normalized against γ-actin and the relative induction of the genes was calculated using the ΔΔCt method. Values represent mean ± SEM of two independent experiments with different donors.

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