Figure 2.

SS2-induced secretion of IL-1β is dependent on suilysin (SLY) in vitro and in vivo. (A,B) Three types of cells, including human monocytic THP-1 cells, mouse peritoneal macrophages (PMs), and BMDMs, were infected with SS2 or its SLY-deficient mutant (∆SLY); multiplicity of infection (MOI, 1) for 16 h. The supernatants were harvested for the measurement of IL-1β (A) and LDH (B). (C–E) BMDMs (1 × 10⁶ cells/ml) were infected with SS2 or ∆SLY (MOI, 1) for 16 h and also treated in parallel with the concentrated supernatant proteins SS2-S/N (200 ng/ml) or ∆SLY-S/N (200 ng/ml) for 16 h after LPS priming for 3 h. Phosphate buffer saline (PBS) and LPS plus adenosine triphosphate (ATP) were used as the negative control and positive control, respectively. The supernatants were collected for IL-1β (C) and IL-6 (E) by ELISA and LDH assay (D). (F) Immunoblotting was performed, and the culture supernatants were measured for IL-1β p17 and Caspase1 p10, and the cell lysates were analyzed for proIL-1β p31 and pro-Caspase1 p45. (G) 8-week-old C57BL/6J WT mice were injected intraperitoneally (IP) with a higher dose of SS2 or ∆SLY (2 × 10⁹ CFU/mouse in 200 μl PBS) and their survivals were observed every 2 h up to 24 h, with 10 mice in each group. The WT mice treated with SS2 and PBS were considered as the positive and negative control group, respectively. (H) 8-week-old WT C57BL/6J mice were injected IP with a lower dose of SS2 or ∆SLY (2 × 10⁸ CFU in 200 μl PBS). The WT mice treated with SS2 and PBS were considered as the positive and negative control group, respectively. These mice were sacrificed 24 h after injection. Their peritoneal lavage fluids (PLFs) were harvested by rinsing the peritoneal cavity with 500 μl PBS and measured for IL-1β by ELISA (six mice in each group). The data shown in panels (A–E) are the means ± standard deviations from three independent experiments. The data shown in panels (F–H) are from one of two independent experiments. ^*p < 0.05; ^**p < 0.01.