Figure 5.

The effect of L-NAME and SNAP on IL-1β, nitrite production and bacterial replication in BMDMs infected with B. abortus. Cells were preincubated with L-NAME (2 mM) or SNAP (0.5 mM) for 30 min before infection with B. abortus strain 2308 (MOI of 100:1) or stimulation with ultrapure LPS (1 μg/mL) or nigericin (Nig, 20 μM). Prior to stimulation with nigericin, cells were primed with ultrapure LPS for 4 hours. Supernatants were harvested 17h after stimulation, except for LPS/Nig-stimulated cells, whose supernatants were harvested 30 min after stimulation. IL-1β (A and B) was determined by ELISA and nitrite (NO₂⁻, C and D) by Griess reaction. The graphs show results from a single experiment representative of four independent experiments performed in triplicate, with similar results and data are presented as mean ± SD. (E) BMDMs were infected with B. abortus (MOI 100:1) and CFU counting were determined 2 and 24 hours post infection (hpi). The graph shows results from a single experiment representative of three independent experiments performed in quadruplicate, with similar results and data are presented as mean ± SD. (F) Western blot analysis of lysates and supernatants of BMDMs infected with B. abortus, preincubated or not with L-NAME (2 mM) or SNAP (0.5 mM). Abbreviations: NI (non-infected), Ctrl (control), LN (L-NAME), SN (SNAP), CL (cell lysate), SN (supernatant). Image is representative of two independent experiments. Significant differences between infected cells in the presence or absence of L-NAME or SNAP are denoted by two or three asterisks (for p<0.01 and p<0.001, respectively; one-way ANOVA + Bonferroni post-hoc test).