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. 2019 Apr 23;87(5):e00809-18. doi: 10.1128/IAI.00809-18

FIG 3.

FIG 3

VpOmpU induces proinflammatory responses in THP-1 monocytes and RAW 264.7 macrophages. (A and B) Maximum production of TNF-α (A) and IL-6 (B) was observed at 24 h in RAW 264.7 cells and at 4 h and 8 h, respectively, in THP-1 cells in response to VpOmpU. RAW 264.7 and THP-1 cells were treated with 10 μg/ml of recombinant VpOmpU for different times, and supernatants were analyzed for TNF-α (A) and IL-6 (B) production by ELISA. (C and D) A dose-dependent increase in TNF-α (C) and IL-6 (D) production is seen in both RAW 264.7 and THP-1 cells in response to different doses of VpOmpU. Cells were incubated with different doses of recombinant VpOmpU, and following incubation, supernatants were collected and analyzed for TNF-α and IL-6 production. RAW 264.7 cells and THP-1 cells were incubated with VpOmpU for 24 h and 4 h, respectively, for TNF-α production (C) and for 24 h and 8 h, respectively, for IL-6 production (D). (E) Nitric oxide (NO) production was observed in VpOmpU-treated RAW 264.7 cells. RAW 264.7 cells were treated with 10 μg/ml of recombinant VpOmpU and incubated for different times. Supernatants were analyzed for NO (in terms of nitrite), and significant production of NO was observed at 24 h. Similar extents of NO production were observed when RAW 264.7 cells were treated with different doses of recombinant VpOmpU and incubated for 24 h. For panels A to E, LPS (1 μg/ml) was used as a positive control for all of the experiments. (F) Cell viability in response to different doses of VpOmpU was minimally affected in both RAW 264.7 and THP-1 cells. For panels A to F, results are expressed as mean ± SEM from three or four independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05 [versus buffer-treated cells]). (G and H) Comparable production of TNF-α and IL-6 was observed in both RAW 264.7 and THP-1 cells in response to similar doses of purified wild-type VpOmpU (wt-VpOmpU) and recombinant VpOmpU (r-VpOmpU). RAW 264.7 and THP-1 cells were treated with 5 μg/ml of wt-VpOmpU or r-VpOmpU and incubated for 24 h and 4 h, respectively, for TNF-α (G) and for 24 h and 8 h, respectively, for IL-6 (H). Results are expressed as mean ± SEM from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05 [versus wt-VpOmpU-treated cells]).