Figure 4.

Acetate increases the secretion of IL-1β via NLRP3. (A) Fold increase of Nlrp3, Asc and Casp1 mRNAs assessed by RT-PCR from MPI cells pre-treated or not with acetate and stimulated or not with S. pneumoniae for 18 h. Fold increase was calculated over control (Ctrl+Vh). (B, C) Western blot for Caspase 1 from supernatant and pro-IL-1β from cellular lysate of MPI cells pre-treated or not with acetate, and then incubated or not with S. pneumoniae for 24 h. Caspase-1 p20 and pro-IL-1β quantifications were normalized to total lane density (total protein) from stain free gel image. For caspase 1, a positive control was done adding 300 ng/mL of LPS for 4 h, followed by 20 µM of nigericin for 30 min. Western blot for caspase 1 was done in duplicates/triplicate and repeated three times. Graphic shows a pool from the mean of three independent experiments. (D) ELISA for IL-1β from supernatants of MPI cells pre-treated or not with acetate, KCl or CY-09 and then stimulated or not with S. pneumoniae for 24 h. (E) Nitrite concentration assessed by Griess assay from supernatant of MPI cells pre-treated or not with acetate in the presence of absence of CY-09 30 µM and stimulated or not with S. pneumoniae during 48 h. (F) % of intracellular viable bacteria left 6 h post infection of activated MPI cells treated or not with acetate and/or CY-09 30 µM, normalized to vehicle group. Bars showing the mean and error showing the SEM (A–E) and data showing the median (F) of triplicates. Results are representative of three independent experiments. Statistical analysis was done using Two-way ANOVA (A–E) and One-Way ANOVA (F) corrected with Sidak’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).