ATP6V0d2 upregulated by intracellular parasites does not participate in parasite resistance to IFN-γ/LPS-activated macrophages.
A. ATP6V0d2 mRNA expression relative to expression of isoform d1 (ATP6V0d1) in nonsilenced or ATP6V0d2-KD macrophages activated or not with IFN-γ/LPS and infected or not by L. amazonensis for 48 hours. The results are representative of 5 independent experiments. *p< 0.05; ns = nonsignificant. B. Expression of NOS2 (left panel) and arginase (right panel) mRNA after 6 and 72 hours of L. amazonensis infection, respectively, in nonsilenced and ATP6V0d2-KD macrophages activated or not with IFN-γ/LPS. NOS2 and arginase mRNA expression was calculated relative to β-actin mRNA expression. The asterisks indicate statistical significance (p<0.05) between nonsilenced and ATP6V0d2-KD measurements. ns = nonsignificant. The results are representative of 2 independent experiments. C-D. Dynamic quantification of parasite numbers in macrophage cultures recorded by live imaging using image segmentation and automatic counting algorithms. In C, upper panel, images of GFP-expressing macrophages (green) merged with images of DsRed2-expressing parasites (red) at the start (0d00:00) and after 20 hours and 30 minutes (0d:20:30) of image acquisition. In the lower panel, the results of image segmentation processing, which identifies parasites (red spots) and macrophages using a color scale ranging from cyan (noninfected macrophage) to magenta (macrophage sheltering >8 parasites). Bar = 10 μm. In D, the number of macrophages per recorded field (upper graph) and the number of parasites per macrophage (lower graph) were assessed throughout 36-hour live imaging recordings of infected nonsilenced or ATP6V0d2-KD macrophages activated or not with IFN-γ/LPS. The data are represented as the means and standard errors of 8 different microscopic fields per condition. The results are representative of 2 independent experiments. E. Infection indexes obtained from nonsilenced or ATP6V0d2-KD macrophages activated or not with IFN-γ/LPS and infected with L. amazonensis for 72 hours. The results are representative of 3 independent experiments.