Fig. 1.
Nitrite generation by Mtb within primary human macrophages cultured in nonhypoxic levels of oxygen. (A) Accumulation of nitrite in supernatant of macrophages in monolayer culture in 10% oxygen infected with WT (Wt) Mtb (MOI: 5; 5 × 105 bacteria) for the indicated times. Box and whisker plots depict median and 5th-95th percentiles of results from six donors. (B) As in A, but macrophages were uninfected or infected with narG-deficient Mtb (NarG) or WT or complemented (NarGc) strains. Results for uninfected macrophages and those infected with NarG Mtb overlap near zero. Means ± SD from one experiment representative of >10. (C) Lack of accumulation of nitrite in nitrate-depleted medium. Macrophages were infected in the presence (*) or absence of 5 mM nitrate. Means ± SD for one experiment representative of two. (D) As in B, but with macrophages maintained under either 21% or 10% oxygen. Means ± SD for one experiment representative of two. (E) Impact of rifampicin on nitrite production. Where indicated, macrophages were infected with WT Mtb (MOI: 5) for 3 d in 10% oxygen in the presence or absence of rifampicin (1 μg/mL). Rifampicin was added 1 d before (D-1), 1 d after (D1), or 2 d after (D2) infection. Uninfected macrophages were treated with rifampicin for the duration of the experiment. Nitrite was measured on day 3 after infection. Means ± SD, n = 2 experiments. (F) As in B, but using ∼20-fold higher numbers of macrophages grown on Cytodex surface microcarrier beads in 10% oxygen and then infected with Mtb (MOI: 5; 107 bacteria). Means ± SD, n = 2 experiments.