IFN-γ-triggered expression and activity of iNOS partially restricts <i>Toxoplasma</i> propagation in SkMCs.

<p>Primary embryonic SkMCs (pSMC) or C2C12 SkMCs were differentiated to mature myotubes. Differentiated SkMCs and control NIH/3T3 fibroblasts were infected at a parasite-host cell ratio of 2∶1 with transgenic <i>T. gondii</i> RH tachyzoites expressing β-galactosidase as a reporter (C–E), or were left non-infected (A,B). Cells were stimulated or not with 100 U/ml IFN-γ alone or combined with 100 pg/ml TNF. (A) Total RNA was isolated at the indicated time points, mRNA was reverse transcribed, and iNOS or β-actin cDNA was amplified by quantitative real-time PCR. Data represents the mean induction of iNOS mRNA by IFN-γ normalized to β-actin; data is from a representative experiment out of two. (B) Nitrite was measured by the Griess reaction in culture supernatants obtained from non-infected C2C12 (black bars) and non-infected NIH/3T3 cells (cross-hatched bars) at the indicated time points. Data represents means ± S.E.M. from at least 5 independent experiments; significant differences between C2C12 and NIH/3T3 cells are indicated (**<i>p</i><0.01). (C–E) Primary SkMCs (C), C2C12 (D) and NIH/3T3 cells (E) were treated with l-NIL (open bars), or were left untreated (black bars) at 1 hour prior to infection and cytokine stimulation. At different time points after infection/cytokine stimulation, growth of β-galactosidase-expressing <i>T. gondii</i> was quantitated by a colorimetric CPRG assay. The inhibition of parasite growth triggered by IFN-γ alone or in combination with TNF was calculated by subtracting OD570 values of cytokine-stimulated cultures from that of non-stimulated control cultures; bars represent means ± S.E.M. from at least two independent experiments.