Figure 4

CATH-1 inhibits SS2-induced inflammatory response by direct bactericidal activity. Mice peritoneal macrophages were infected with SS2 strain SC19 at a MOI of 5 and 10 in the presence of CATH-1 (1.25 and 2.5 μM) for 2 and 6 h (co-incubation). At 2 and 6 hpi, gentamicin (250 μg/mL) were added and continued to incubate for 22 and 18 h. After 24 h, supernatants were collected to determine cytokine secretion including IL-1β, IL-6, and TNF-α using ELISA (A and C). Meanwhile, at 2 hpi, partial cells were directly lysed by 0.1% Triton X-100 to quantify adherent bacteria and gentamicin was added to another partial cells for 30 min before lysis to quantify intracellular bacteria (B). For post-incubation studies, macrophages were infected with SS2 strain SC19 for 2 h and then CATH-1 (1.25 and 2.5 μM) and gentamicin (250 μg/mL) were added respectively for 2 h. At 4 hpi, gentamicin (250 μg/mL) was added and continued to culture for 20 h. Finally, supernatants were collected to determine cytokine secretion including IL-1β, IL-6, and TNF-α using ELISA (D); In addition, at 4 hpi, cells were lysed to quantify intracellular bacteria (E). Data are represented as mean ± SEM of three independent experiments of triplicate samples per experiment. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ns, no significance.